Duck embryonic derived stem cell lines for the production of viral vaccines

ABSTRACT

The present invention relates to the development and manufacturing of viral vaccines. In particular, the invention relates to the field of industrial production of viral vectors and vaccines, more in particular to the use of avian embryonic stem cells, preferably the EBx® cell line derived from duck embryonic stem cells, for the production of viral vectors and viruses. The invention is particularly useful for the industrial production of viral vaccines to prevent viral infection of humans and animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/069,423, filed Nov. 1, 2013, which is a divisional of U.S.application Ser. No. 12/597,486, filed on Oct. 23, 2009, which is anational stage filing under 35 U.S.C. §371 of International ApplicationNo. PCT/EP2008/054912, filed on Apr. 23, 2008, which claims the priorityof European Application No. 07300979.7, filed Apr. 24, 2007, the entirecontents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the development and manufacturing ofviral vaccines. In particular, the invention relates to the field ofindustrial production of viral vectors and vaccines, more specificallyto the use of duck cell lines derived from embryonic stem cells that arefree of avian endogenous retrovirus, for the production of viral vectorsand viruses. The invention is particularly useful for the industrialproduction of viral vaccines to prevent viral infection of humans andanimals.

BACKGROUND

Vaccines effectively reduce and prevent death and disease from manyviral infections such as for example flu, measles, mumps, smallpox,yellow fever.

Many viral vaccines are currently produced on embryonated chicken eggsor on primary chicken embryo fibroblasts isolated from chicken embryos.However, vaccine production occasionally has been complicated byinadvertent contamination with adventitious agents that may haveoriginated from avian cell substrates used to propagate vaccine strains.Indeed reverse-transcriptase (RT) activity, an indication of thepresence of retroviruses, was detected in chick cell-derived live,attenuated vaccines including those produced by European and USmanufacturers for yellow fever, Measles and Mumps (Hussain et al., 2003,J. Virol 77:1105-1111; Johnson et Heneine, 2001, J. Virol.,75:3605-3612). Investigations of the origin of RT activity in thosevaccines found evidence of particles containing RNA from endogenousavian leucosis virus (ALV-E) and endogenous avian virus (EAV) (Johnsonet Heneine, 2001, J. Virol 75:3605-3612; Tsang et al., 1999, J. Virol73:5843-5851; Weissmahr et al., 1997, J. Virol 71:3005-3012).

Both ALV-E and EAV are members of endogenous retrovirus families presentin the chicken germ line. ALV-E are expressed from ev loci, which areinheritable proviral elements. Based on their envelope sequences, ALV-Eare differentiated from ALV subgroups A to D and J which are exogenouslyacquired infections. While exogenous ALVs cause several neoplasticdiseases, such as myocarditis and osteopetrosis in infected chickens,ALV-E are not known to be pathogenic to chickens. The lack of oncogenicpotential with ALV-E infections may be attributed to the absence of botha viral oncogene and enhancer activity in the endogenous long terminalrepeat (LTR). More than 20 different ev loci have been identified inWhite Leghorn chickens (ev-1 to ev-22). Ev loci designations areassigned in the order discovered and are phenotypically categorized withregard to the gene products they express and their capacity to generateinfectious particles. ALV-E phenotypes conferred by ev loci range fromstructurally and enzymatically complete infectious particles tostructurally or enzymatically (RT-) defective to no detectable viralprotein expression. Most ev loci are structurally incomplete andtherefore do not encode all sequences necessary for production ofinfectious virus particles. Chicken strain, named ev-0, has beenobtained by breeding to be resistant to ALV-E. Line-0 chickens arelacking ev loci (i.e ev-0) but EAV proviral sequences are present in thegenome line 0 chickens (Dunwiddie and Faras, 1985, Proc Natl. Acad. ScUSA, 82: 5097-5101).

Little is known about the EAV family, which is distinct from but relatedto ALV family. EAV elements are present in at least 50 copies perchicken genome. However, none of the known EAV sequences representslull-length and intact retroviral genomes and no infectious EAV isolateshave been yet identified. However EAV have been shown to be highlyexpressed in embryonic cells derived from the avian genus, gallus.Weissmahr et al. (1997, J. Virol 71:3005-3012) have shown that particlesfrom the EAV endogenous retrovirus family are most likely responsiblefor a large portion of the particles-associated RT activity found in thesupernatants of cultured chick embryo fibroblasts.

The risk of inadvertent transmission is particularly high for liveattenuated virus vaccine since they cannot be subjected to aninactivation procedure and most of them are injected into human, thusby-passing non-specific immune protection mechanisms. Thus, to ensuresafety of vaccines for animal and human use, the cell substrates forvaccine production have now to be tested for the presence ofreplication-competent retroviruses that could be passed to animal orhuman hosts during immunization (WHO technical reports Series, 1994).

On the other hands, embryonated chicken eggs and primary chicken embryofibroblasts production systems are associated with several seriouslimitations, including:

-   -   a lengthy, cumbersome and resource-consuming manufacturing        process that requires the procurement and quality control of        large quantities of eggs or CEFs for each individual production        campaign;    -   the need in many cases to use costly specific pathogen free        (SPF) chicken embryos;    -   the risks of insufficient supply of eggs in cases of epidemic        infections in donor chicken flocks;    -   the inflationist costs associated with the use of bovine sera        originating from BSE-exempt countries;    -   the inability to use eggs for the propagation of viruses that        are highly virulent and lethal to chickens.

There is therefore an urgent need to improve on the current viralvaccines production technologies based on eggs or chicken-embryonicfibroblasts. The development of cell-culture platforms as an alternativeto the eggs and CEF production systems for the manufacture of viralvaccines is likely the most rapid and promising solution to overcomecurrent vaccines production bottlenecks and time constrains. Moreover,the use of cell lines for manufacture of viral vaccines, instead of eggor CEF platforms, would have the additional following advantages inconnection with the safety of the vaccine: no antibiotic additivespresent in the vaccine formulation; no toxic preservatives (such asthiomersal) needed; reduced endotoxin levels, no egg allergy issue; norisk of adventitious agent/BSE by cell culture in protein and serum freemedia; higher purity of virus vaccine preparation.

Examples of cell lines for the production of viral vaccines are MDCK(cells derived from the kidney of Madin-Darby dog), PerC6 (cells derivedfrom human embryonic retinal cells genetically modified by inserting theE1 genes from the human adenovirus type 5) developed by CRUCELL(Netherland)). VERO (cells derived from epithelial cells of kidney fromAfrican green monkey (Cercopithecus aethiops) isolate at the ChibaUniversity in Chiba, Japan), BHK21 (Cells immortalized from baby hamsterkidney cells). None of the cell lines available fulfil all the medical,regulatory and industrial requirements. For example, most of these celllines are tumorigenic and there are important regulatory concern aboutthe use of tumorigenic cells for the production of human vaccines;therefore, today the regulatory authorities are reluctant to approvetumorigenic cell substrates to produce mass vaccines. In addition, someof these cell lines are anchorage-dependant, which constitutes a serioushurdle for the industrial scaling-up of the vaccine production.

Therefore, there is a need to develop anchorage-independent cell lines,free of replication competent of retroviruses, that are non-tumorigenicand industrially compliant, which is susceptible to infection with awide range of viruses. This is the purpose of the instant invention.

Thus, the inventor has taken advantage of its expertise in avian biologyand in avian embryonic stem (ES) cells to undertake the development ofnovel stable duck cell lines that enables the efficient replication of alarge group of human and veterinarian vaccines and vaccine candidates.By adapting a proprietary process (see WO 03/076601 and WO 05/007840),the inventor was able to generate a series of well characterized anddocumented duck cell lines (i.e the dEBx® cells) that are derived fromduck ES cells, with no steps of genetic, chemical or viralimmortalization and that do not produce replication-competentretroviruses in culture.

DESCRIPTION

The instant invention provides a process for obtaining continuousdiploid avian cell lines, named EBx, derived from avian embryonic stemcells (ES), wherein said avian cell lines do not producereplication-competent endogenous retrovirus particles.

The cell lines of the invention are “continuous” because they have thecharacteristics to be cultured in vitro over an extended period of time.Advantageously, the cells of the invention are capable of proliferatingfor at least 50 generations, at least 75 generations, at least 100generations, at least 125 generations, at least 150 generations, atleast 175 generations, at least 200 generations, at least 250generations. The 250 generations do not constitute a time limit becausethe cells obtained are still alive and can still be passaged foradditional passages. Without to be bond by a theory, it is postulatedthat the cells of the invention can be cultured “continuously” as longas telomerase is expressed by the cells. Indeed, it is assumed that thehigh level of telomerase expression of avian cells of the invention isresponsible for genetic stability (i.e avian cells of the invention arediploid) and the continuous cell growth.

By “passage” it is meant the transfer of transplantation of cells, withor without dilution, from one culture vessel to another. It isunderstood that any time cells are transferred from one vessel toanother, a certain portion of the cells may be lost and therefore,dilution of cells, whether deliberate or not, may occur. This term issynonymous with the term ‘subculture’. The passage number is the numberof times the cells in the culture, that grow either in suspension or inadherence, have been sub-cultured or passed in a new vessel. This termis not synonymous with population doubling or generation which is thetime needed by a cell population to replicate one time; that is to say,roughly the time for each cells of a population to replicate. Forexample, Avian ES cells of step a) of the invention have a populationdoubling time (PDT) of around >40 hours. The avian EBx cells of theinvention have a PDT of around <30 hours; usually for EBx® cells, thereis one passage every 3 generations.

By “diploid”, it is mean that cells of the invention have two copies(2n) of each chromosome, usually one from the mother and one from thefather.

The fact that avian EBx® cell lines of the invention are continuous anddiploid (i.e genetically stable) constitutes a remarkable and uniquefeature because these terms are usually antagonist. Thus, cancer cellsand/or immortalized cells obtained by chemical, physical (U.Virradiation, X-ray or g-irradiation, . . . ) or genetic modification(virus transformation, oncogenes overexpression, . . . ) are continuouscells because they are able to replicate indefinitely into culture, butthey are not genetically stable because they display polyploidkaryotypes. On the other hand, primary cells such as chicken embryonicfibroblasts, MRC5, WI38 which are non-transformed cells, are notcontinuous because they have a finite life-span after few generation,but they are genetically stable (i.e diploid) cells.

In the present invention, the terms “cell line” and “cells” will be usedindistinctly.

The term “avian, “bird”, “aves” or “ava” as used herein is intended tohave the same meaning, and will be used indistinctly. “Birds” refer toany species, subspecies or race of organism of the taxonomic class<<ava>>. In a preferred embodiment, “birds” refer to any animal of thetaxonomix order:

-   -   “Anseriformes” (i.e duck, goose, swan and allies). The order        Anseriformes contains about 150 species of birds in three        families: the Anhimidae (the screamers), Anseranatidae (the        Magpie-goose), and the Anatidae, which includes over 140 species        of waterfowl, among them the ducks, geese, and swans. All        species in the order are highly adapted for an aquatic existence        at the water surface. All are web-footed for efficient swimming        (although some have subsequently become mainly terrestrial).    -   “Galliformes” (i.e chicken, quails, turkey, pheasant and        allies). The Galliformes is an order of birds containing the        chicken, turkeys, quails and pheasants. About 256 species are        found worldwide.    -   “Columbiformes” (i.e Pigeon and allies). The bird order        Columbiformes includes the very widespread doves and pigeons.

In the instant invention, by the term “endogenous retroviral particle”or “endogenous retrovirus particle”, terms that could be usedindistinctively, it is meant a retroviral particle or retrovirus encodedby and/or expressed from ALV-E or EAV proviral sequences present in someavian cell genomes. In the birds, ALV-E proviral sequences are known tobe present in the genome of domestic chicken (except Line-0 chicken),red jungle fowl and Ringneck Pheasant. In the birds, EAV proviralsequences are known to be present in all genus gallus that includesdomestic chicken, Line-0 chicken, red jungle fowl, green jungle fowl,grey jungle fowl, Ceylonese jungle fowl and allies) (see Resnick et al.,1990, J. Virol., 64:4640-4653).

According to a preferred embodiment, the bird of the invention areselected among the birds that does not comprises ALV-E and EAV proviralsequences in its genome. A man skilled in the art is able to determinewhether ALV-E and EAV sequences are present in a bird genome (Johnsonand Heneine, 2001; Weissmahr et al., 1996). Preferably the bird isselected in the group comprising Anserilormes (i.e duck, goose, swan),turkeys, quails, Japanese quail, Guinea fowl, Pea Fowl. Therefore, cellsderived from such bird do not produce replication-competent endogenousALV-E and/or EAV particles. In a preferred embodiment, the bird of thepresent invention is selected among the group comprising ducks, geese,swans, turkeys, quails and Japanese quails, Guinea Fowls and Pea Fowls.According to a more preferred embodiment, the bird is a duck, morepreferably a Pekin or Muscovy ducks. According to a more preferredembodiment, the bird is a Pekin duck. Therefore, the instant inventionprovides a process for obtaining continuous diploid duck cell linesderived from embryonic stem cells (ES), wherein said duck cell lines donot produce replication-competent endogenous retrovirus particles.

According to a second preferred embodiment, the bird of the inventionare selected among the birds that does not comprises complete ALV-Eproviral sequences in its genome but eventually EAV proviral sequences.A man skilled in the art is able to determine whether partial orcomplete ALV-E and EAV sequences are present in a bird genome (Johnsonand Heneine, 2001). Several chicken strains have been selected bybreeding that do not contain complete ALV-E proviral sequences (i.e:ev-0 strain) and therefore do not produce infectious ALV-Eretroparticles, such as:

-   -   Line 0 domestic chicken of East Lansing USDA poultry stock        (ELL-0 strain). The East Lansing Line-0 chickens do not contain        any endogenous viral (ev) loci related to ALV (Dunwiddie and        Faras, 1985).    -   Lines DE and PE11 from Institut National de la Recherche        Agronomique (Domaine de Magneraud, Surgeres, France).

Therefore, cells derived from ev-0 birds do not producereplication-competent endogenous ALV-E particles. According to apreferred embodiment, the bird is an ev-0 domestic chicken (GallusGallus subspecies domesticus), preferably selected among ELL-0, DE andPE11.

Usually, ev-0 chickens still contain EAV proviral sequence but so far noinfectious EAV isolates have been identified. Therefore, the instantinvention provides a process for obtaining continuous diploid chickencell lines derived from embryonic stem cells (ES) of ev-0 chickenstrains, wherein said ev-0 chicken cell lines do not producereplication-competent endogenous retrovirus particles.

According to a third embodiment, the bird of the invention are selectedamong the birds that comprise complete and/or incomplete ALV-E and EAVproviral sequences in its genome but that are unable to producereplication competent ALV-E and EAV retroparticles. A man skilled in theart is able to determine whether ALV-E and/or EAV infectious and/ornon-infectious retroparticles are produced from a bird cells (Johnsonand Heneine, 2001; Weissmahr et al., 1996). Preferably the bird isselected in the group comprising specific pathogen free (SPF) chicken,preferably from Valo strain (Lohman) or Line 22 (SPAFAS).

By “replication-competent” it is meant that the endogenous retroviralparticles are infectious, that is to say that such retroviral particulesare able to infect and to replicate in avian cells of the invention.

The process of establishment of continuous diploid avian cell lines,named EBx®, of the invention comprises two steps:

-   -   a) isolation, culture and expansion of embryonic stem cells from        birds that do not contain complete endogenous proviral        sequences, or a fragment thereof, susceptible to produce        replication competent endogenous retroviral particles, more        specifically EAV and/or ALV-E proviral sequences or a fragment        thereof, in a complete culture medium containing all the factors        allowing their growth and in presence of a feeder layer and        supplemented with animal serum; optionally, said complete        culture medium may comprise additives, such as additional        amino-acids (i.e glutamine, non essential amino acids . . . ),        sodium pyruvate, beta-mercaptoethanol, vitamins, protein        hydrolyzate of non-animal origin (i.e yeastolate, plant        hydrolyzates (soy, wheat, . . . );    -   b) passage by modifying the culture medium so as to obtain a        total withdrawal of said factors, said feeder layer and said        serum, and optionally said additives, and further obtaining        adherent or suspension avian cell lines, named EBx®, that do not        produce replication-competent endogenous retrovirus particles,        capable of proliferating over a long period of time, in a basal        medium in the absence of exogenous growth factors, feeder layer        and animal serum.

The modification of the culture medium of step b) of the process ofestablishment EBx® cell lines, so as to obtain progressive or totalwithdrawal of growth factors, serum and feeder layer, can be madesimultaneously, successively or separately. The sequence of the weaningof the culture medium may be chosen among:

-   -   feeder layer/serum/growth factors;    -   feeder layer/growth factors/serum;    -   serum/growth factors/feeder layer;    -   serum/feeder layer/growth factors;    -   growth lactors/serum/leeder layer;    -   growth factors/feeder layer/serum.

In a preferred embodiment, the sequence of the weaning is growthfactors/feeder layer/serum. In a preferred embodiment, the withdrawal ofadditives such as sodium pyruvate, non essential amino acids (NNEA),vitamins, yeastolate are performed after the weaning of feeder layer andbefore the weaning of serum. Preferably, the withdrawal of yeastolate isperformed after the withdrawal of sodium pyruvate, NNEA and vitamins.

According to a preferred embodiment, the avian embryonic stem cellsaccording to step a) of the invention are collected from avian embryo atoviposition, that is to say when the egg is laid. According to Sellieret al. (2006, J. Appl. Poult. Res., 15:219-228), oviposition correspondsto the following development stages according to Eyal-Giladi'sclassification (EYAL-GILADI's classification: EYAL-GILADI and KOCHAN,1976, <<From cleavage to primitive streack formation: a complementarynormal table and a new look at the first stages of the development inthe chick>>. “General Morphology” Dev. Biol. 49:321-337):

-   -   Muscovy duck (also called Barbari duck): stage VII    -   Guinea fowl: stage VII-VIII    -   Turkey: stage VII-VIII    -   Pekin duck: stage VIII    -   Chicken: Stage X    -   Japanese Quail: stage XI    -   Goose: stage XI.

Preferably, the duck embryonic stem (ES) cells of step a) are obtainedby dissociating Pekin duck embryo(s) at around stage VIII (oviposition)of Eyal-Giladi's classification. If the laid egg collected atoviposition is not enough developed to collect embryonic stem cells, thelaid egg may be further incubated between several hours (overnight) toone to two days to mature the embryo. According to a second embodimentthe duck embryonic stem (ES) cells of step a) is from a Muscovy duck. Atoviposition, Muscovy duck is not enough mature because it is aroundstage VII, therefore, the egg is incubated overnight to mature the eggup to stage VIII to X of Eyal-Giladi's classification.

Preferably, the chicken embryonic stem (ES) cells, preferably from ev-Ochicken strain, of step a) is obtained by dissociating embryo(s) ataround stage X (oviposition) of Eyal-Giladi's classification.

Alternatively, the avian embryonic stem cells according to step a) ofthe invention are collected from embryo before oviposition. The mainlimitations encountered before oviposition is the fact that the egg hasto be surgically removed from hens and that the amount of ES cells perembryo is less important. Moreover at very early stages of avian embryodevelopment, ES cells are not well individualized rendering difficult invitro culture of ES cells. A man skilled in the Art will be able todefine the timeframe prior egg laying that allows to collect avian EScells.

Alternatively, the avian embryonic stem cells according to step a) ofthe invention may be collected from avian embryo after oviposition up tohatching. However, avian embryonic stem cells will progressively enterinto differentiation to generate differentiated tissues: therefore, itis preferred to collect avian ES not to long after the lay. A manskilled in the Art will be able to define the timeframe after egg layingthat allows to collect avian embryonic stem cells.

According to another embodiment, the cells of step a) are a populationof embryonic stem cells enriched in primordial germ cells (PGC). Morepreferably, the avian ES cells of step a) are purified PGCs. In avianspecies, Primordial Germ Cells arise from the central region of theblastoderm (Ginsburg and Eyal-Giladi, 1987 Development 101(2):209-19;Karagenc et al, 1996 Dev Genet 19(4):290-301; Petitte et al, 1997Poultry Sci. 76(8):1084-92). Then they move to an anterior,extra-embryonic site, the germinal crescent until collected by thevasculature between 2.5 and 5 days of embryonic development to reach thegerminal ridge. They colonize the germinal ridge where they eventuallydifferentiate into oocytes or spermatocytes (Nieuwkoop and Sutasurya,1979. The Migration of the primordial germ cells. In: Primordial germcell in Chordates. London: Cambridge University Press p 113-127).Methods for isolation of PGCs from donor avian embryos have beenreported in the literature and can easily be performed by one skilled inthe art (See, e.g. JP924997 published Sep. 7, 1993 Pub. No 05-227947;Chang et al. 1992. Cell Biol. Int. 19(2): 143-149; Naito et al. 1994Mol. Reprod. Dev. 39: 153-161; Yasuda et al. 1992. J. Reprod. Fert. 96:521-528; Chang et al. 1992 Cell Biol. Int. Reporter 16(9): 853-857).According to an embodiment, PGCs are collected from embryonic bloodcollected from the dorsal aorta of a chicken embryo at stage 12-14 ofHamburger & Hamilton's classification (Hamburger & Hamilton 1951 Aseries of normal stages in the development of chick embryo. J. Morphol.88: 49-92). In another preferred embodiment, PGCs were collected fromthe germinal crescent by mechanical dissection of chicken embryo or fromthe gonads. However, as discussed above, others methods for isolatingPGCs are known and can alternatively be used.

These avian embryonic stem cells are characterized by a slow doublingtime comprises between 48 to 72 hours in culture at 39° C.

Without to be bound by a theory, the defined cell culture conditions ofavian ES cells followed by the progressive weaning in grow factors,feeder layer, additives and serum, allow to adapt and select cells thatmaintain most of the desirable feature of ES cells (stability ofkaryotype, indefinite proliferation, expression of ES markers) but inaddition display industrial-friendly characteristics like growth insuspension up to high cell densities in serum-free medium. Telomeraseconstitutes one of the most important ES markers. Due to the sustainedand maintained telomerase expression over the cell passages, EBx® cellare continuous (i.e immortal) but in addition are genetically stable(i.e diploid).

More specifically, the present invention provides a process forobtaining continuous diploid avian cell lines derived from ES cells,wherein said avian cell lines do not produce replication competentendogenous retroviral particles, said process comprising the followingsteps of:

-   -   a) isolating bird embryo(s), preferably from duck or from ev-0        chicken, at a developmental stage comprises from around stage VI        of Eyal-Giladi's classification (EYAL-GILADI's classification:        EYAL-GILADI and KOCHAN, 1976, <<From cleavage to primitive        streack formation: a complementary normal table and a new look        at the first stages of the development in the chick>>. “General        Morphology” Dev. Biol., 49:321-337) and before hatching,        preferably around oviposition, wherein the genome of said bird        does not contain endogenous proviral sequences susceptible to        produce replication competent endogenous retroviral particles;    -   b) suspending avian embryonic stem (ES) cells obtained by        dissociating embryo(s) of step a) in a basal culture medium        supplemented with:        -   Insulin Growth factor 1 (IGF-1) and Ciliary Neurotrophic            factor (CNTF);        -   animal serum; and        -   optionally, growth factors selected in the group comprising            interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), Stem            cell Factor (SCF) and Fibroblast Growth Factor (FGF);    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the ES cells for at        least one passage;    -   d) optionally withdrawing all the growth factors selected from        the group comprising IL-6, IL-6R, SCF, FGF from the culture        medium over a range of several passages from 1 to around 15        passages, preferably from 3 to around 15 passages and further        culturing the avian ES cells for at least one passage.        Preferably, the withdrawing of all the growth factors selected        from the group comprising IL-6, IL-6R, SCF, FGF from the culture        medium is performed simultaneously over one passage. Usually,        the withdrawing of IL-6, IL-6R, SCF, FGF is performed at around        passage 10 to 15;    -   e) withdrawing IGF-1 and CNTF from the culture medium and        further culturing the avian ES cells for at least one passage.        Preferably, the withdrawing of the growth factors selected from        the group comprising IGF-1 and CNTF from the culture medium is        performed simultaneously, over one passage. Usually, the        withdrawing of IGF-1 and CNTF is performed at around passage No        15 to No 25. Alternatively, the withdrawing of IGF-1 and CNTF is        performed by progressive decreasing over several passages (at        least 2 passages and approximately up to 15 passages);    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer alter several passages, and further culturing the cells;    -   g) optionally, progressively decreasing the concentration of        additives in the culture medium so as to obtain a total        withdrawal of additives after at least one passage; and,    -   h) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages; and,    -   i) obtaining adherent avian cell lines, named EBx®, derived from        ES cells capable of proliferating in a basal medium in the        absence of growth factors, feeder layer optionally without        animal serum and additives, and wherein said continuous diploid        avian cell lines do not produce replication-competent endogenous        retrovirus particles;    -   j) optionally, further adapting said adherent avian EBx® cell        lines to suspension culture conditions. The step of adaptation        of cell culture to suspension can take place all along the        process of establishment of EBx® cells. For example, with duck        EBx® cells derived from Muscovy embryonic stem cells, the cells        were adapted to the growth in suspension prior feeder layer        withdrawal. For duck EB® cells (EB24, EB26, EB66) derived from        Pekin duck, the cells were adapted to the growth in suspension        prior animal serum withdrawal.    -   k) Optionally further subcloning said avian EBx® cells, for        example by limit dilution.

In a preferred embodiment, the present invention relates to a processfor obtaining continuous diploid avian cell lines, named EBx®, derivedfrom avian embryonic stem cells (ES), wherein said avian cell lines donot produce replication-competent endogenous retrovirus particles, andsaid process comprising the steps of:

-   -   a) isolating bird embryo(s) at a developmental stage around        oviposition, wherein the genome of said bird does not contain        endogenous proviral sequences susceptible to produce replication        competent endogenous retroviral particles;    -   b) suspending avian embryonic stem (ES) cells obtained by        dissociating embryo(s) of step a) in a basal culture medium        supplemented with at least:        -   Insulin Growth factor 1 (IGF-1) and Ciliary Neurotrophic            factor (CNTF); and        -   mammalian serum such as foetal bovine serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the ES cells for at        least one passage;    -   e) withdrawing IGF-1 and CNTF from the culture medium, and        further culturing the cells for at least one passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer after several passages, and further culturing the cells;    -   g) progressively decreasing the concentration of said mammalian        serum in the culture medium so as to obtain a total withdrawal        of mammalian serum after several passages and:    -   h) obtaining adherent avian EBx® cell lines derived from ES        cells capable of proliferating in a basal medium in the absence        of growth factors, feeder layer and mammalian serum, and wherein        said continuous diploid avian cell lines do not produce        replication-competent endogenous retrovirus particles;    -   i) optionally, further adapting adherent avian EBx® cell lines        to suspension culture conditions, preferably by promoting the        growth as suspension, more preferably by transferring the        adherent avian EBx® cell lines obtained in step h) in another        support having lower attachment characteristic than the initial        support (i.e. such as Ultra Low attachment support).

Step j) of adapting adherent avian EBx® cell lines to suspension cultureconditions, when carried out, can be effected in another preferredembodiment before the step g) of progressively decreasing theconcentration of mammalian serum in the culture medium.

In another preferred embodiment, the basal culture medium in step b) ofthe process for obtaining continuous diploid avian cell lines accordingto the present invention, is further supplemented with a growth factorselected in the group comprising interleukin 6 (IL-6), interleukin 6receptor (IL-6R), Stem cell Factor (SCF) and Fibroblast Growth Factor(FGF), and the said process further comprises a step d) of:

-   -   d) optionally withdrawing all the growth factors selected from        the group comprising IL-6, IL-6R, SCF, FGF from the culture        medium and further culturing the ES cells for at least one        passage.

In a more preferred embodiment, when step d) is carried out, the step e)of withdrawing IGF-1 and CNTF from the culture medium, is effected afterthe step d) of withdrawing all the growth factors selected from thegroup comprising IL-6, IL-6R, SCF, FGF from the culture medium.

According to the invention, “basal culture medium” meant a culturemedium with a classical media formulation that allows, by itself, atleast cells survival, and even better, cell growth. Examples of basalmedia are BME (basal Eagle Medium), MEM (minimum Eagle Medium), medium199, DMEM (Dulbecco's modified Eagle Medium), GMEM (Glasgow modifiedEagle medium), DMEM-HamF12, Ham-F12 and Ham-F10, Iscove's ModifiedDulbecco's medium, MacCoy's 5A medium, RPMI 1640, GTM3. Basal mediumcomprises inorganic salts (for examples: CaCl₂, KCl, NaCl, NaHCO₃,NaH₂PO₄, MgSO₄, . . . ), amino-acids, vitamins (thiamine, riboflavin,folic acid, D-Ca panthothenate, . . . ) and others components such asglucose, beta-mercapto-ethanol, sodium pyruvate. Preferably basal mediumis a synthetic medium. Table 1 gives the composition of DMEM/HAM F12:

TABLE 1 DMEM-HAM F12 Formulation (mg/l) Inorganic Salts Calcium Chlorideanhydrous 116.60 Ferric(III)-Nitrate•9H₂O 0.05 Ferric(II)-Sulphate•7H₂O0.417 Potassium Chloride 311.80 Cupric(II)-Sulphate•5H₂O 0.0013Magnesium Chloride•6H₂O 61.20 Magnesium Sulphate anhydrous 48.84 SodiumChloride 6996.00 Sodium Dihydrogen Phosphate•H₂O 62.50 Di-SodiumDihydrogen Phosphate anhydrous 71.02 Zinc Sulphate•7H₂O 0.432 SodiumHydrogen Carbonate 1200.00 Amino Acids L-Alanine 4.45 L-Arginine•HCl147.50 L-Asparagine•H₂O 7.50 L-Aspartic Acid 6.65 L-Cystine•HCl•H₂O31.29 L-Cysteine•2HCl 17.56 L-Glutamic Acid 7.35 L-Glutamine in E15-813365.00 Glycine 18.75 L-Histidine•HCl•H₂O 31.48 L-Isoleucine 54.47L-Leucine 59.05 L-Lysine•HCl 91.25 L-Methionine 17.24 L-Phenylalanine35.48 L-Proline 17.25 L-Serine 26.25 L-Threonine 53.45 L-Tryptophan 9.02L-Tyrosine 38.70 L-Valine 52.85 Vitamins D(+)-Biotin 0.0035 D-CalciumPantothenate 2.24 Choline Chloride 8.98 Folic Acid 2.65 Myo-Inositol12.60 Nicotinamide 2.02 Pyridoxal•HCl 2.00 Pyridoxine•HCl 0.031Riboflavin 0.219 Thiamine•HCl 2.17 Thymidine 0.365 Vitamin B12 0.68Other Components D-Glucose anhydrous 3151.00 Hypoxanthine 2.10DL-68-Lipoic Acid 0.105 Linoleic Acid 0.042 Phenol Red 8.10Putrescine•2HCl 0.081 Sodium Pyruvate 55.00

In addition, basal medium of the invention may be complemented withadditives selected in the following group:

-   -   0.1 to 5 mM L-glutamine, preferably between 2 to 3 mM        L-Glutamine;    -   0.05 to 2 mM sodium pyruvate, preferably between 0.1 mM to 1 mM        sodium pyruvate;    -   0.1 to 2.5% non-essential amino-acids, preferably around 1%        non-essential amino-acids;    -   0.1 to 2.5% vitamins, preferably around 1% vitamins    -   0.05 to 5 mM beta-mercapto-ethanol, preferably around 0.16 mM        beta-mercapto-ethanol;    -   protein hydrolyzate of non-animal origin.

For the establishment of duck EBx® cells of the invention, the basalmedium is preferably complemented with protein hydrolyzate of non-animalorigin. Protein hydrolyzates of non-animal origin are selected from thegroup consisting bacteria tryptone, yeast tryptone, plant hydrolyzates,such as soy hydrolyzates, or a mixture thereof. In a preferredembodiment, the protein hydrolyzates of non-animal origin is yeasthydrolyzate. The term “hydrolyzate” includes an enzymatic digest of soypeptone or yeast extract. The hydrolysate can be obtained from aplurality of soy peptone or yeast extract preparations, respectively,which can be further enzymatically digested (for example, by papain),and/or formed by autolysis, thermolysis and/or plasmolysis. Hydrolysatesalso may be obtained commercially, such as Yeastolate, Hy-Soy, Hy-Yeast412 and Hi-Yeast 444, from sources such as SAFC BioSciences (formerlyJRH) (Lenaxa, Kans.), Quest International (Norwich, N.Y.), OrganoTechnieS.A. (France) or Deutsche Hefewerke GmbH (Germany). Sources of yeastextracts also are disclosed in WO 98/15614. Sources of yeast extractsand soy hydrolysates also are disclosed in WO00/03000. The hydrolysatesused in media of the invention are preferably purified from a crudefraction, because impurities which could interfere with efficientcultivation are preferably eliminated during this purification, therebyimproving the consistency of the hydrolysate. Purification can be byultrafiltration or Sephadex chromatography (for example, with SephadexG25 or Sephadex G10 or equivalent materials), ion-exchangechromatography, affinity chromatography, size exclusion chromatographyor “reversed-phase” chromatography. Preferably, purification isperformed by ultrafiltration utilizing a 10 kDa cut-off filter. Theseprocesses are known in the field. Using these methods, fractions can beselected which contain soy or yeast hydrolysate of defined molecularweight. Preferably, the average molecular weights of the soy and yeasthydrolysates are preferably between about 220 and 375 daltons.Preferably, yeast hydrolyzate is present in the cell culture medium.Yeast hydrolyzate 50× (around 200 g/I) obtained for example fromSAFC-BIOSCIENCES (Ref 58902C) is present in the cell culture medium at afinal concentration comprises between around 0.1× to 2×, preferablyaround 0.5× to around 1× into the culture medium. Soy hydrolyzate mayalso be added to the cell culture medium. Soy hydrolyzate 50× obtainedfor example from SAFC-BIOSCIENCES (Ref 58903C) is added at a finalconcentration comprises between around 0.1× to 2×, preferably around 1×into the culture medium. Alternatively a mixture of soy hydrolyzate andyeast hydrolyzate may be added to the cell culture medium as describedin US2004/0077086.

According to a preferred basal medium of the invention is DMEM-HamF12that are complemented with 2 mM L-glutamin, 1 mM sodium pyruvate, 1%non-essential amino-acids, vitamins 1%, 0.16 mM beta-mercapto-ethanol,and optionally with 1× yeast hydrolyzate.

By “complete culture medium”, it is meant a basal culture mediumcomplemented or not, preferably a basal synthetic medium, supplementedwith at least one growth factor and animal serum. Example of completeculture medium is described in WO 03/076601, WO 05/007840, EP 787180,U.S. Pat. No. 6,114,168, U.S. Pat. No. 5,340,740, U.S. Pat. No.6,656,479, U.S. Pat. No. 5,830,510 and in Pain et al. (1996, Development122:2339-2348). Alternatively, the complete culture medium may aconditioned medium, preferably BRL conditioned medium. By way ofexample, BRL conditioned media is prepared according to art-recognizedtechniques, such as described by Smith and Hooper (1987, Dev. Biol.121:1-9). BRL cells are available from ATCC accession number CRL-1442.Conditioned medium may be supplemented with exogenous growth factors andanimal serum as described below.

The term “growth factors” as used herein meant growth factor necessaryfor the survival and the growth of the undifferentiated avian ES cellsin culture in a basal culture medium. It is possible to schematicallydistinguish two families of growth factors: the cytokines and thetrophic factors. The cytokines are mainly cytokines whose action isthrough a receptor which is associated with the gp130 protein. Thus,leukemia inhibitory factor (LIF), interleukin 11, interleukin 6,interleukin 6 receptor, Ciliary Neurotrophic factor (CNTF), oncostatinand cardiotrophin have a similar mode of action with the recruitment atthe level of the receptor of a specific chain and the combination of thelatter with the gp130 protein in monomeric or sometimes hetero-dimericform. The trophic factors are mainly Stem cell Factor (SCF), InsulinGrowth factor 1 (IGF-1) and Fibroblast Growth Factor (FGF), preferablybasic FGF (bFGF) or human FGF (hFGF).

The complete culture medium according to the invention comprises basalculture medium, preferably basal synthetic medium, and at least onecytokine whose action is through a receptor which is associated with thegp130 protein and/or at least one trophic factors. Preferably, thecomplete culture medium according to the invention comprises basalmedium and at least one growth factor selected in the group consistingof Leukemia Inhibitory factor (LIF), oncostatin, cardiotrophin, InsulinGrowth factor 1 (IGF-1), Ciliary Neurotrophic factor (CNTF), Interleukin6 (IL-6), interleukin 6 receptor (IL-6R), Stem cell Factor (SCF),Fibroblast Growth Factor (FGF), interleukin 11 (IL-11). According to afirst preferred embodiment, the complete culture medium is basal mediumsupplemented with animal serum and with at least IGF-1 and CNTF.According to a second preferred embodiment, the complete culture mediumis basal medium supplemented with animal serum and at least IGF-1, CNTF,IL-6 and IL-6R. According to a third preferred embodiment, the completeculture medium is basal medium supplemented with animal serum and atleast IGF-1, CNTF, IL-6, IL-6R, SCF, FGF. According to anotherembodiment, the complete culture medium is a conditioned culture mediumcomprising growth factors (i.e expressed by BRL or STO cells forexample) and optionally supplemented with at least one exogenous growthfactors selected in the group comprising: LIF, IGF-1, CNTF, IL-6, IL-6R,SCF, FGF, IL-11. The concentration of growth factors IGF-1, CNTF, IL-6,IL-6R, SCF, FGF, IL-11 in the basal medium or in the conditioned culturemedium is comprised between about 0.01 to 10 ng/ml, preferably, 0.1 to 5ng/ml, and more preferably about 1 ng/ml.

The culture medium of the invention may also comprise in additionantibiotics, such as for example, gentamicine, penicilline andstreptomycine, to prevent bacterial contamination. Antibiotics may beadded to the culture medium at the early passages of ES cells culture.For example, gentamycin at a final concentration of 10 ng/ml, penicillinat a final concentration of 100 U/ml and streptomycin at a finalconcentration of 100 μg/ml may be added to the culture medium. In apreferred embodiment, no antibiotics is added to the culture mediumduring the late steps of process of establishment of continuous diploidavian cell lines of the invention.

During the process of establishment of avian embryonic stem cells of theinvention, the cells are cultured on a layer of feeder cells. Morepreferably, feeder cells are animal cells or cell lines cultured for thepurpose of culturing avian ES cells. Alternatively, the feeder cells canbe substituted with extra-cellular matrix plus bound growth factors.Feeder matrix will thereafter refers to either feeder cells orextra-cellular matrix. A feeder matrix as used herein is constructed inaccordance with procedures known in the art. As noted above, it ispreferred that the feeder matrix be preconditioned. By the term“preconditioned” it is meant that the feeder matrix is cultured in thepresence of media for a period of time prior to the depositing of cellsoriginating from the blastoderm disk fertilized avian eggs in contactwith the feeder matrix, e.g. a time sufficient to initiate and establishproduction of, for example, growth factors or other factors by thefeeder matrix; usually a feeder matrix is preconditioned by culturingthe feeder matrix by itself for one to two days prior to the depositingof cells originating from the blastoderm disk fertilized avian eggs incontact with the feeder matrix. The feeder cells preferably comprisesmouse fibroblast cells. STO fibroblasts are preferred, but primaryfibroblasts are also suitable. Also while the present invention has beendescribed with respect to the use of mouse cell feeder matrices, it iscontemplated that feeder matrices comprising cells from other murinespecies (e.g. rat); other mammalian species (e.g; ungulate, bovine,porcine species); or avian species (e.g. Gallinacea, chicken, turkey,duck, goose, quail, pheasant) may also be used. In another embodiment,feeder cells of the invention may be transfected with expressionvector(s) allowing for example the constitutive expression of growthfactors such as avian SCF in STO cells. Thus, this “feeder” produces thefactor in a form which is soluble and/or attached in the plasma membraneof the cells. Thus, the culturing process of the present invention mayoptionally comprise establishing a monolayer of feeder cells. Feedercells are mitotically inactivated using standard techniques. Forexample, the feeder cells may be exposed to X or gamma radiation (e.g.4000 Reads of gamma radiation) or may be treated with Mitomycin C (e.g.10 μg/ml for 2-3 hours). Procedures for mitotically inactivating cellsare also detailed in the information typically sent with cells from theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209 (e.g. STO feeder cells are available under ATCCaccession number 1503). Mono-layers may optionally be cultured to about80% confluency, preferably to about 90% confluency, and more preferablyabout 100% confluency. While configuration of the feeder cells as amonolayer is the preferred configuration for the culture, any suitableconfiguration is contemplated to be within the scope of the presentinvention. Thus, for example, layers, mono-layers, clusters, aggregatesor other associations or groupings of feeder cells are contemplated tofall within the scope of the present invention and are particularlycontemplated to fall with the meaning of the term “matrix”.

The culture medium of the invention is supplemented with animal serum.The animal serum preferably used is fetal animal serum. Fetal bovineserum is preferred. Also while the present invention has been describedwith respect to the use of fetal bovine serum, it is contemplated thatanimal serum comprising serum from other animal species (e.g. chicken,horse, porcine, ungulate, etc.) may also be used. The finalconcentration of animal serum in the culture medium is comprises betweenapproximately 1 to 25%, preferably between 5% to 20%, more preferablybetween 8% and 12%. In the preferred embodiment, the final concentrationof animal serum in the culture medium is approximately 10%. According toa preferred embodiment, the culture medium comprises approximately 10%of fetal calf serum.

In a first preferred embodiment, the bird of the present invention isselected in the Order of Anseriformes, and is preferably a duck, morepreferably a Pekin Duck, and more preferably Pekin duck strain M14 orGL30. According to a second preferred embodiment, the bird of thepresent invention is a Muscovy duck. Therefore, the instant inventionprovides a first process for obtaining continuous diploid duck celllines derived from embryonic stem cells (ES), wherein said duck celllines do not produce replication-competent endogenous retrovirusparticles, and said process is comprising the steps of:

-   -   a) isolating duck embryo(s) at oviposition (i.e egg laying), or        slightly prior or after oviposition. Optionally, the egg may be        incubated, usually overnight, to mature (i.e Muscovy duck);    -   b) suspending duck embryonic stem (ES) cells obtained by        dissociating embryo(s) of step a) in a basal culture medium        supplemented with Insulin Growth factor 1 (IGF-1), Ciliary        Neurotrophic factor (CNTF), interleukin 6 (IL-6), interleukin 6        receptor (IL-6R), Stem cell Factor (SCF) and Fibroblast Growth        Factor (FGF) and animal serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the duck ES cells        for at least 1 passage;    -   d) withdrawing all the growth factors selected from the group        comprising IGF-1, CNTF, IL-6, IL-6R, SCF, FGF from the culture        medium over a range of 1 to around 15 passages, preferably        simultaneously over one passage, and further culturing the duck        ES cells for at least one passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium, from a passage to another, so as to obtain a        total withdrawal of feeder layer after several passages,        preferably, after around 5 to around 25 passages, and further        culturing the cells;    -   g) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages; and:    -   h) obtaining adherent duck cell lines derived from ES cells,        named duck EBx®, capable of proliferating in a basal medium in        the absence of growth factors, feeder layer optionally without        animal serum, and wherein said continuous diploid duck cell        lines do not produce replication-competent endogenous retrovirus        particles;    -   i) optionally, further adapting adherent duck cell lines to        suspension culture conditions. Additives to basal medium are        withdrawn during the process, and preferably between steps f)        and g) or between steps g) and h).

The animal serum concentration at step b) is preferably of 5 to 10%. Theconcentration of with Insulin Growth factor 1 (IGF-1), CiliaryNeurotrophic factor (CNTF), interleukin 6 (IL-6), interleukin 6 receptor(IL-6R), Stem cell Factor (SCF) and Fibroblast Growth Factor (FGF) arepreferably of about 1 ng/ml.

The instant invention also provides a second process for obtainingcontinuous diploid duck cell lines derived from embryonic stem cells(ES), wherein said duck cell lines do not produce replication-competentendogenous retrovirus particles, and said process is comprising thesteps of:

-   -   a) isolating duck embryo(s) at oviposition (i.e egg laying), or        slightly prior or after oviposition. Optionally, the egg may be        incubated, usually overnight, to mature (i.e Muscovy duck);    -   b) suspending duck embryonic stem (ES) cells obtained by        dissociating embryo(s) of step a) in a basal culture medium        supplemented with IGF-1, CNTF, IL-6, IL-6R, SCF and FGF and        animal serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the duck ES cells        for at least 1 passage;    -   d) withdrawing all the growth factors selected from the group        comprising IL-6, IL-6R, SCF, FGF from the culture medium over a        range of 1 to around 15 passages, preferably simultaneously over        one passage, and further culturing the duck ES cells for at        least one passage;    -   e) withdrawing the growth factors IGF-1 and CNTF from the        culture medium over a range of 1 to around 15 passages,        preferably simultaneously over one passage, and further        culturing the duck ES cells for at least one passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer after several passages, preferably, after around 5 to        around 25 passages, and further culturing the cells;    -   g) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages; and    -   h) obtaining adherent duck cell lines derived from ES cells,        named duck EBx®, capable of proliferating in a basal medium in        the absence of growth factors, feeder layer optionally without        animal serum, and wherein said continuous diploid duck cell        lines do not produce replication-competent endogenous retrovirus        particles;    -   i) optionally, further adapting adherent duck cell lines to        suspension culture conditions. Additives to basal medium are        withdrawn during the process, and preferably between steps f)        and g) or between steps g) and h).

The animal serum concentration at step b) is preferably of 5 to 10%. Theconcentration of IGF-1, CNTF, IL-6, IL-6R, SCF and FGF are preferably ofabout 1 ng/ml.

The instant invention also provides a third process for obtainingcontinuous diploid duck cell lines derived from embryonic stem cells(ES), wherein said duck cell lines do not produce replication-competentendogenous retrovirus particles, and said process is comprising thesteps of:

-   -   a) isolating duck embryo(s) at oviposition (i.e egg laying), or        slightly prior or after oviposition. Optionally, the egg may be        incubated, usually overnight, to mature (i.e Muscovy duck);    -   b) suspending duck embryonic stem (ES) cells obtained by        dissociating embryo(s) of step a) in a basal culture medium        supplemented with Insulin Growth factor 1 (IGF-1), and Ciliary        Neurotrophic factor (CNTF) and animal serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the duck ES cells        for at least 1 passage;    -   d) withdrawing the growth factors IGF-1 and CNTF from the        culture medium over a range of 1 to around 15 passages,        preferably simultaneously over one passage, and further        culturing the duck ES cells for at least one passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer after several passages, preferably, after around 5 to        around 25 passages, and further culturing the cells; Removal of        additives?    -   g) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages; and,    -   h) obtaining adherent duck cell lines derived from ES cells,        named duck EBx®, capable of proliferating in a basal medium in        the absence of growth factors, feeder layer optionally without        animal serum, and wherein said continuous diploid avian cell        lines do not produce replication-competent endogenous retrovirus        particles;    -   i) optionally, further adapting adherent duck EBx® cell lines to        suspension culture conditions. Additives to basal medium are        withdrawn during the process, and preferably between steps f)        and g) or between steps g) and h).

The animal serum concentration at step b) is preferably of 5 to 10%. Theconcentration of with IGF-1 and CNTF are preferably of about 1 ng/ml.

Once adherent or suspension duck cell lines have been obtained, theprocess of the invention may also comprises the additional step ofadapting, duck EBx® cells to the growth in cell culture medium withoutprotein hydrolyzate of non-animal origin, such as yeast hydrolyzates.

Preferably, duck EBx® cell lines of the invention do not display reversetranscriptase activity by O-PERT analysis. Moreover, noreplication-competent endogenous retrovirus particles is produced byduck EBx® cells as demonstrated by co-culture experiments of duck EBx®cells of the invention with ALV replication competent cells, such asquail QT6 cells or chicken DF1 cells. In addition, transmissionelectronic microscopy (TEM) analysis also demonstrate the absence ofreplication-competent endogenous retrovirus particles in duck EBx®cells. Preferably, the duck EBx® cell line of the invention is selectedamong duck EB24, duck EB26 and duck EB66 as described hereinafter.

In another preferred embodiment, the bird of the present invention isselected in the Order of Galliformes and more preferably is a chicken,preferably an a ev-0 domestic chicken (Gallus Gallus subspeciesdomesticus). Therefore, the instant invention provides a process forobtaining continuous diploid ev-0 domestic chicken cell lines derivedfrom embryonic stem cells (ES), wherein said ev-0 domestic chicken celllines do not produce replication-competent endogenous ALV-E retrovirusparticles, and said process is comprising the steps of:

-   -   a) isolating ev-0 domestic chicken embryo(s) at oviposition (i.e        egg laying) or slightly prior or after oviposition:    -   b) suspending ev-0 domestic chicken embryonic stem (ES) cells        obtained by dissociating embryo(s) of step a) in a basal culture        medium supplemented with IGF-1, CNTF, IL-6, IL-6R, SCF and FGF        and animal serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the ev-0 domestic        chicken ES cells for at least 1 passage;    -   d) withdrawing all the growth factors selected from the group        comprising IGF-1, CNTF, IL-6, IL-6R, SCF, FGF from the culture        medium over a range of 1 to around 15 passages, preferably        simultaneously over one passage, and further culturing the        chicken ES cells for at least one passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer after several passages, preferably, after around 5 to        around 25 passages, and further culturing the cells;    -   g) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages and:    -   h) obtaining adherent ev-0 domestic chicken cell lines derived        from ES cells, named EBx ev-0, capable of proliferating in a        basal medium in the absence of growth factors, feeder layer        optionally without animal serum, and wherein said continuous        diploid avian cell lines do not produce replication-competent        endogenous ALV-E retrovirus particles;    -   i) optionally, further adapting adherent avian cell lines EBx        ev-0 to suspension culture conditions.    -   Additives to basal medium are withdrawn during the process, and        preferably between steps f) and g) or between steps g) and h).

The animal serum concentration at step b) is preferably of 5 to 10%. Theconcentration of IGF-1, CNTF, IL-6, IL-6R, SCF and FGF are preferably ofabout 1 ng/ml.

The instant invention also provides a second process for obtainingcontinuous diploid ev-0 domestic chicken cell lines derived fromembryonic stem cells (ES), wherein said ev-0 domestic chicken cell linesdo not produce replication-competent endogenous ALV-E retrovirusparticles, and said process is comprising the steps of:

-   -   a) isolating ev-0 domestic chicken embryo(s) at oviposition (i.e        egg laying) or slightly prior or after oviposition;    -   b) suspending ev-0 domestic chicken embryonic stem (ES) cells        obtained by dissociating embryo(s) of step a) in a basal culture        medium supplemented with IGF-1, CNTF, IL-6, IL-6R, SCF and FGF        and animal serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the ev-0 domestic        chicken ES cells for at least 1 passage;    -   d) withdrawing all the growth factors selected from the group        comprising IL-6, IL-6R, SCF, FGF from the culture medium over a        range of 1 to around 15 passages, preferably simultaneously over        one passage, and further culturing the chicken ES cells for at        least one passage;    -   e) withdrawing the growth factors IGF-1 and CNTF from the        culture medium over a range of 1 to around 15 passages,        preferably simultaneously over one passage, and further        culturing the ev-0 domestic chicken ES cells for at least one        passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer after several passages, preferably, after around 5 to        around 45 passages, and further culturing the cells;    -   g) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages and:    -   h) obtaining adherent ev-0 domestic chicken cell lines derived        from ES cells capable of proliferating in a basal medium in the        absence of growth factors, feeder layer optionally without        animal serum, and wherein said continuous diploid ev-0 domestic        chicken cell lines, named chicken EBx®, do not produce        replication-competent endogenous retrovirus particles;    -   i) optionally, further adapting adherent ev-0 domestic chicken        cell lines to suspension culture conditions.    -   Additives to basal medium are withdrawn during the process, and        preferably between steps f) and g) or between steps g) and h).

The animal serum concentration at step b) is preferably of 5 to 10%. Theconcentration of with IGF-1, CNTF, IL-6, IL-6R, SCF and FGF arepreferably of about 1 ng/ml.

The instant invention also provides a third process for obtainingcontinuous diploid ev-0 domestic chicken cell lines derived fromembryonic stem cells (ES), wherein said ev-0 domestic chicken cell linesdo not produce replication-competent endogenous ALV-E retrovirusparticles, and said process is comprising the steps of:

-   -   a) isolating ev-0 domestic chicken embryo(s) at oviposition (i.e        egg laying) or slightly prior or after oviposition;    -   b) suspending ev-0 domestic chicken embryonic stem (ES) cells        obtained by dissociating embryo(s) of step a) in a basal culture        medium supplemented with IGF-1 and CNTF and animal serum;    -   c) seeding the suspension of ES cells obtained in step b) on a        layer of feeder cells and further culturing the ev-0 domestic        chicken ES cells for at least 1 passage;    -   d) withdrawing the growth factors IGF-1 and CNTF from the        culture medium over a range of 1 to around 15 passages,        preferably simultaneously over one passage, and further        culturing the ev-0 domestic chicken ES cells for at least one        passage;    -   f) progressively decreasing the concentration of feeder cells in        the culture medium so as to obtain a total withdrawal of feeder        layer after several passages, preferably, after around 5 to        around 45 passages, and further culturing the cells;    -   g) optionally, progressively decreasing the concentration of        animal serum in the culture medium so as to obtain a total        withdrawal of animal serum after several passages; and,    -   h) obtaining adherent ev-0 domestic chicken cell lines derived        from ES cells, named chicken EBx® ev-0, capable of proliferating        in a basal medium in the absence of growth factors, feeder layer        optionally without animal serum, and wherein said continuous        diploid chicken cell lines do not produce replication-competent        endogenous retrovirus particles;    -   i) optionally, further adapting adherent ev-0 domestic chicken        cell lines to suspension culture conditions.    -   Additives to basal medium are withdrawn during the process, and        preferably between steps f) and g) or between steps g) and h).

The animal serum concentration at step b) is preferably of 5 to 10%. Theconcentration of with IGF-1 and CNTF are preferably of about 1 ng/ml.

In another preferred embodiment, the bird of the present invention is adomestic chicken (Gallus Gallus subspecies domesticus) obtained from aspecific-pathogen-free (SPF) flock. More preferably, the chicken strainis White-Leghorn. SPF chicken eggs has been screened for the absence ofknown chicken bacterial pathogens and viruses, including thereticuloendotheliosis virus (REV) and the avian exogenous leucosis virus(ALV-A, ALV-B, ALV-C, ALV-D, ALV-J). The SPF egg of the invention mayVALO eggs from LOHMANN (Cuxhaven, Germany) or L22 eggs from CHARLESRIVER (Spafas). Therefore, the instant invention also provides processesfor obtaining continuous diploid chicken cell lines derived fromembryonic stem cells (ES) obtained from SPF chicken eggs, like describedwith ev-0 chicken eggs. Preferably, the chicken EBx® cell line obtainedfrom SPF eggs is EBv13.

Chicken EBx® cell lines of the invention may display reversetranscriptase activity by Q-PERT analysis, but without producingreplication-competent endogenous retrovirus particles. The absence ofreplication-competent endogenous retrovirus particles may bedemonstrated by co-culture experiments of chicken EBx® ev-0 cells of theinvention with ALV replication competent cells, such as quail QT6 cellsor chicken DF1 cells. In addition, the absence of endogenous retrovirusparticles in chicken EBx® ev-0 cells may be also demonstrate by TEM.

Body temperature of bird is usually around 39° C. Therefore, theprocesses of the invention may also comprise the additional step ofdecreasing the cell culture temperature to 37° C. in order to adapt theavian cell lines of the invention to grow at 37° C. Preferably, thetemperature adaptation is performed after feeder depletion and priorserum depletion. Alternatively, the temperature adaptation is performedafter the serum depletion step or after the step of adapting the celllines to suspension culture.

The established lines EBx® of the invention have the characteristic togrow either as adherent cells or as suspension cells in a culture mediumfree of exogenous growth factors and animal serum and without feedercells. Different techniques can be used alone or in combination to adaptcells to suspension culture, among them:

-   -   Adherent cells are seeded at high cell density, slightly above        cell confluence to force the cells to go into suspension;    -   Adherent cells are seeded in a cell culture medium with a low        animal serum concentration;    -   Adherent cells are seeded onto cell culture vessels made of        plastic that do not allow cell adhesion or a weak cell adhesion,        such as bacterial dishes and plates and ultra-low attachment        plates developed by companies like Corning (tissue culture        dishes & plates Ref. 3262, 3473, 3471, 3474; Flasks Ref. 3814 .        . . ) or Sarstedt (Flask ref 831810502 . . . );    -   Adherent cells are seeded on vessel and cultured under agitation        (Approx. 50 rpm).

The EBx® cells, preferably duck EBx® and chicken EBx® ev-0, can be invitro cultured over a considerable period of time. Advantageously, theadherent or anchorage-independent (i.e “suspension) EBx® cells obtainedby the process of the invention are capable to proliferate for at least50 generation, at least 75 generation, at least 100 generation, at least125 generation, at least 150 generation, at least 175 generation, atleast 200 generation, at least 250 generation. The expression “line” isunderstood to mean any population of cells capable of proliferatingindefinitely in culture in vitro while retaining to a greater or lesserdegree the same morphological and phenotypic characteristics. Clones maybe obtained, for example by limit dilution, from EBx® cells of theinvention. These clones are cells which are genetically identical to thecell from which they are derived by division.

The present invention also relates to the continuous diploid avian celllines, named EBx®, obtainable by the process of the invention, said EBx®being small, round (i.e diameter around 10 μm), individualized cellswith a doubling time of around 30 hours or less at 37° C. or 39° C. Theavian EBx® cells, preferably the duck EBx® or chicken EBx® ev-0, expressan embryonic stem cell phenotype with the following characteristics:

-   -   a high nucleo-cytoplasmic ratio,    -   an endogenous telomerase activity,    -   optionally, they may express one or more additional ES markers        such as alkaline phosphatase, SSEA-1, EMA-1, ENS1 markers.    -   A doubling time shorter than the doubling time of the avian ES        cells of step a) of the process of the invention (48 h to 72 h        at 39° C.), of about 30 hours or less (preferably 24 hours) at        37° C.

Said cells do not produce replication competent endogenous retrovirusparticles.

The avian EBx® cell lines of the invention are capable of proliferatingindefinitely in a basal medium, in particular in a medium such as SAFCExcell media, DMEM, GMEM, DMEM-HamF12 or McCoy, free of exogenous growthfactors, serum and/or inactivated feeder layer, optionally complementedwith various additives commonly used by persons skilled in the artExamples of additives are non-essential amino acids, vitamins, sodiumpyruvate and antibiotics. Duck EBx® cells of the invention have theremarkable feature to grow in a basal culture medium that is notcomplemented with glutamine.

The present invention also relates to a cell culture medium to maintainpluri- or multipotent avian embryonic stem cells, preferably pluri- ormultipotent duck embryonic stem (ES) cells, into culture in anundifferentiated state. According to a preferred embodiment, the presentinvention relates to cell culture medium for duck embryonic stem cellscomprising a basal culture medium, supplemented with animal serum andsupplemented with at least IGF-1 and CNTF. According to a secondpreferred embodiment, the present invention relates to cell culturemedium for duck embryonic stem cells comprising a basal culture mediumsupplemented with animal serum and supplemented with at least IGF-1,CNTF, 11-6, II-6R. According to a third preferred embodiment, thepresent invention relates to a cell culture medium for duck embryonicstem cells comprising a basal culture medium supplemented with animalserum and supplemented with at least IGF-1, CNTF, 11-6, 11-6R, SCF andFGF. Said media are sufficient for the maintenance of said duck ES cellsinto culture for at least 7 days, preferably for at least 20 days,preferably for at least 100 days in an undifferentiated state. Saidculture media of the invention may further comprise optionally at leastone compound selected in the group comprising Interleukin-11,cardiotrophin, oncostatin and leukaemia inhibitory factor (LIF).Preferably, said culture media further comprise protein hydrolyzate ofnon-animal origin as previously described; more preferably it is yeasthydrolyzate at 1× concentration. The culture medium of avian (preferablyduck) ES cells of the invention may further comprise a layer of feedercells.

The instant invention also provide a sustained duck ES cell cultureconsisting essentially of undifferentiated duck ES cells expressing stemcell phenotype with the following characteristics:

-   -   a high nucleo-cytoplasmic ratio,    -   an endogenous telomerase activity,    -   optionally, duck ES cells may express one or more additional ES        markers such as alkaline phosphatase, SSEA-1, EMA-1, ENS1        markers.    -   A doubling time of about around than 40 hours at 37° or 39° C.

Said undifferentiated duck cells according to the invention are capableof maintaining said stem cell phenotype when grown on feeder cells in acell culture medium for duck embryonic stem cells as previouslydescribed. Said undifferentiated duck cells are useful to producechimeric or transgenic ducks.

Therefore, the present invention also relates to a method of obtainingchimeric duck, said method comprising the steps of:

-   -   a) introducing a sustained duck ES cell culture as described        above into the sub-germinal cavity of a recipient duck embryo;        and    -   b) incubating the embryo obtained in step a) to hatch as a        duckling;    -   c) selecting said chimeric duckling comprising heterologous        cells having colonized said duckling.

The present invention also relates to a method of obtaining geneticallymodified chimeric duck, comprising the steps of:

-   -   a) introducing a genetically modified duck ES cells as described        above into the sub-germinal cavity of a recipient duck embryo;        and    -   b) incubating the embryo obtained in step a) to hatch as a        duckling;    -   c) selecting said chimeric duckling comprising genetically        modified heterologous cells having colonized said duckling.

The present invention also relates to a method of obtaining a progeny ofsaid chimeric duckling wherein said method comprises the followingsteps:

-   -   a) allowing the selected chimeric duckling obtained at steps c)        to mature as an adult bird;    -   b) breeding said adult bird having heterologous cells herein,        thereby producing a bird progeny;    -   c) selecting the birds of interest in the progeny.

The invention may comprise the additional step of expressing anheterologous polypeptide encoded by an expression vector comprised insaid genetically modified duck ES cells. Preferably, the heterologouspolypeptide is delivered into biological fluid of duck, such as blood,sperm, urine, or the white of a developing avian egg produced by afemale of the genetically modified duck.

The EBx® cells of the invention have all the above mentionedcharacteristics and are useful for the production of biologics such asviral vaccines and recombinant peptides and proteins.

The instant invention also provide a process of replicating a virus inthe continuous diploid avian EBx® cell lines of the invention. Morepreferably, the invention provides a process of replicating a virus inthe continuous diploid avian EBx® cell lines of the invention,preferably duck or chicken EBx® cell lines, that comprise the steps of:

-   -   infecting an avian EBx® cell culture with a virus of interest;        said avian EBx® cells being preferably cultured in animal serum        free medium;    -   culture of infected avian EBx® cells in order to replicate said        virus;    -   harvest the virus in cell culture supernatant and/or inside said        cells.

According to a preferred embodiment, said process comprises the stepsof:

-   -   a) proliferating said avian EBx® in a cultivation vessel, in        suspension, in a serum-free medium No 1;    -   b) infecting said cells with the selected virus when the cell        density is of at least 1.5 million cells/ml;    -   c) optionally, shortly before infection, simultaneously to        infection, or shortly after infection adding to the cell culture        serum-free medium No 2; and    -   d) further culturing said infected cells in order to allow virus        replication; and    -   e) optionally, harvesting said virus.

Said process of the invention may comprise the additional step of addingproteolytic enzyme in the culture medium in conditions that allow viruspropagation. The proteolytic enzyme is selected from the groupconsisting of trypsin, chymotrypsine, thermolysine, pepsine,pancreatine, papaTne, pronase, subtilisine A, elastase, furine andcarboxypeptidase. According to a preferred embodiment, the enzyme istrypsin. Preferably, the proteolytic enzyme is a recombinant proteinproduced on a procaryotic host or on plants (i.e: trypzean). Theproteolytic enzyme may added before, during and/or after the virusinfection. Preferably, the addition of proteolytic enzyme is performedafter virus infection. The addition of proteolytic enzyme in the culturemedium may be performed one time per day, more than one time per day, orless than one time per day until the virus harvest.

The term “virus” as used herein includes not only naturally occurringviruses but also attenuated viruses, reassortant viruses, vaccinestrains, as well as recombinant viruses and viral vectors derivedthereof. The virus of the invention are preferably selected from thegroup comprising poxviruses, orthomyxoviruses, paramyxoviruses, herpesviruses, hepadnaviruses, adenoviruses, parvoviruses, reoviruses,circoviruses, coronaviruses, flaviviruses, togaviruses, birnavrirusesand retroviruses.

In a preferred embodiment, the viruses, the related viral vectors, viralparticles and viral vaccines belong to the family of poxviridae, andmore preferably to the chordopoxviridae. In one embodiment, the virus orthe related viral vectors, viral particles and viral vaccines are apoxvirus, preferably an avipoxvirus selected among fowlpox virus (i.eTROVAC), canarypox virus (i.e ALVAC), juncopox virus, mynahpox virus,pigeonpox virus, psittacinepox virus, quailpoxvirus, sparrowpoxvirus,starling poxvirus, turkeypox virus. According to another preferredembodiment, the virus is a vaccinia virus selected among Lister-Elstreevaccinia virus strain, modified vaccinia virus such as Modified Vacciniavirus Ankara (MVA) which can be obtained from ATCC (ATCC NumberVR-1508), NYVAC (Tartaglia et al., 1992, Virology, 188:217-232), LC16m8(Sugimoto et Yamanouchi, 1994, Vaccine, 12:675-681), CV178 (Kempe etal., 1968, Pediatrics 42:980-985) and other recombinant ornon-recombinant vaccinia virus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family ofortho-myxoviridae, in particular influenza virus. The influenza virus isselected from the group consisting of human influenza virus, avianinfluenza virus, equine influenza virus, swine influenza virus, felineinfluenza virus. Influenza virus is preferably selected in strains A, Band C. Among strains A, one can recite viruses with different subtypesof haemagglutinin and neuraminidase, such as without limitation H1N1,H2N2, H3N2, H4N2, H4N6, H5N1, H5N2, H7N7 et H9N2. Among H1N1 strains,one can recite A/Porto Rico/8/34, A/New Caledonia/20/99,A/Beijing/262/95, A/Johannesburg/282/96, A/Texas/36/91, A/Singapore,A/Solomon Islands/03/2006. Among strains H3N2, one can reciteA/Panama/2007/99, A/Moscow/10/99, A/Johannesburg/33/94,A/Wisconsin/10/04. Among B strains, one can recite without limitationB/Porto Rico/8/34, B/Johannesburg/5/99, B/Vienna/1/99, B/Ann Arbor/1/86,B/Memphis/1/93, B/Harbin/7/94, N/Shandong/7/97, B/Hong Kong/330/01,B/Yamanashi/166/98, B/Jiangsu/10/03, B/Malaysia. The influenza Virus ofthe invention is selected among wild type virus, primary viral isolateobtained from infected individual, recombinant virus, attenuated virus,temperature sensitive virus, low-temperature adapted virus, reassortantvirus, reverse genetic engineered virus. When the virus of the inventionis influenza virus, the process of the invention comprises theadditional step of adding proteolytic enzyme in the culture medium inconditions that allow virus propagation. According to a preferredembodiment, the enzyme is trypsin. The final concentration of trypsin incell culture medium is comprises between around 0.01 μg/ml up to 10μg/ml. More preferably, the final concentration of trypsin in cellculture medium is comprised between 0.01 to 10 usp/ml (usp: USpharmacopea unit) preferably around between 0.05 to 2 usp/ml, morepreferably around between 0.3 to 1 usp/ml and more preferably around0.75 usp/ml.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family ofparamyxoviridae. Preferably the virus is a naturally occurringparamyxovirus or a recombinant paramyxovirus selected in the groupcomprising measles virus, mumps virus, rubella virus, Sendai virus,Respiratory Syncythial virus (RSV), human para-influenza types I andIII, Rinderpest virus, canine distemper virus, Newcastle disease virus,duck para-influenza virus. According to preferred embodiment, the virusis measles virus or a recombinant measles virus. According to anotherpreferred embodiment, the virus is Newcastle Disease virus (NDV) or arecombinant NDV. Example of NDV strain is LaSota strain. When the virusof the invention is NDV, the process of the invention comprisespreferably the additional step of adding proteolytic enzyme in theculture medium in conditions that allow virus propagation. According toa preferred embodiment, the enzyme is trypsin. The final concentrationof trypsin in cell culture medium is comprises between around 0.01 μg/mlup to 10 μg/ml. More preferably, the final concentration of trypsin incell culture medium is comprised between 0.01 to 10 usp/ml (usp: USpharmacopea unit) preferably around between 0.3 to 1 usp/ml, morepreferably around between 0.4 to 0.75 usp/ml. Interestingly, the EBx®cell lines of the invention that may grow in adherence are useful toperform virus titration, and preferably NDV titration, on a plaqueassay. Indeed, unlike CEFs and chicken DF1 fibroblasts for which is wasnot possible to observe any cytopathic effects, virus growth in EBx®cells leads to the formation of characteristic giant cells. In addition,NDV viral particles may be determined by haemagglutination assay.Therefore, the invention also pertain to the use of EBx® cells of theinvention for the titration of virus, such as NDV virus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of togaviridae.Preferably the virus is a naturally occurring alphavirus or arecombinant alphavirus selected in the group comprising Sinbis virus,Semliki forest virus, O'nyong'nyong virus, Chikungunya virus, Mayarovirus, Ross river virus, Eastern equine encephalitis virus, WesternEquine encephalitis virus, Venezuelan Equine encephalitis virus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of herpesviridae.Preferably the virus is a naturally occurring Marek Disease virus or arecombinant Marek Disease virus. The Marek Disease virus (MDV) ispreferably selected among the license vaccine strains of MDV such as:FC126 (HTV), SB-1, 301B/1, CV1988 Clone C, CV1988/C/R6, CV1988/Rispens,R2/23 (Md11/75).

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of hepadnaviridae.Preferably the virus is a naturally occurring naturally occurringhepadnavirus or a recombinant hepadnavirus, preferably selected amongavian and human hepadnavirus. The avian hepadnavirus is preferablyselected among the group consisting of duck hepatitis B virus (DHBV),heron hepatitis B virus (HHBV) and snow goose (SGHBV).

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of birnaviridae,in particular Infectious Bursal Disease virus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of flaviviridae,in particular Dengue virus, Japanese encephalitis virus and West Nilevirus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of coronaviridae,in particular Infectious Bronchitis virus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of circoviridae,in particular Chicken Anemia virus.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of retroviridae.Preferably the virus is a naturally occurring retrovirus selected amongreticulo-endotheliosis virus, duck infectious anemia virus, suck spleennecrosis virus, or a recombinant retrovirus thereof.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of parvoviridae.Preferably the virus is a naturally occurring parvovirus such as duckparvovirus or a recombinant parvovirus thereof.

In another preferred embodiment, the viruses, the related viral vectors,the viral particles and vaccines belong to the family of adenoviridae.Preferably the virus is a naturally occurring adenovirus preferablyselected among fowl adenovirus, goose adenovirus, duck adenovirus andpigeon adenovirus or a recombinant adenovirus thereof. Examples of Fowladenovirus are Fowl adenovirus 1 (CELO), Fowl adenovirus 5 (340), Fowladenovirus 4 (KR95), Fowl adenovirus 10 (CFA20), Fowl adenovirus 2(P7-A), Fowl adenovirus 3 (75), Fowl adenovirus 9 (A2-A), Fowladenovirus 11 (380), Fowl adenovirus 6 (CR119), Fowl adenovirus 7(YR36), Fowl adenovirus 8a (TR59) Fowl adenovirus 8b (764) and Egg DropSyndrome virus. Examples of Goose adenovirus are Goose adenovirus 1,Goose adenovirus 2, Goose adenovirus 3. Example of Duck adenovirus isDuck adenovirus 2. Example of Pigeon adenovirus is Pigeon adenovirus 1.

Recombinant viruses include but are not limited to viral vectorscomprising a heterologous gene. In some embodiments, a helperfunction(s) for replication of the viruses is provided by the host cellEBx®, a helper virus, or a helper plasmid. Representative vectorsinclude but are not limited to those that will infect avian or mammaliancells.

The instant invention also relates to the use of EBx® cells of theinvention to replicate intracellular bacteria such as Chlamydia,Rickettsia or Coxiella.

The EBx® cells of the invention may also be used to produce recombinantproteins and peptides. The invention also relates to a method ofproduction of recombinant proteins and peptides, that include the stepsof: (i) genetically modifying the EBx® cells of the invention bytransient or stable transfection of an expression vector; (ii)optionally, selecting EBx® cells expressing said recombinant proteins orpeptides; (iii) and purification of said peptides or proteins. Peptidesand proteins produced in EBx@cells are also included in the presentinvention.

The cultivation vessel of the invention is more preferably selectedamong continuous stirred tank bioreactor, Wave™ Bioreactor, Bello™bioreactor, spinner flask, flask and a cell factory. Typically, cellsare scaled-up from a master or working cell bank vial through varioussizes of T-flasks, roller bottles or Wave™ Bioreactor and, preferably,finally to bioreactors. The resulting cell suspension is then typicallyfed into a seed production bioreactor (typically 20-30 L volume) forfurther cultivation, and in some embodiments, to a larger productionbioreactor (typically 150-180 L volume and above). The ratio of volumeof the second (larger) bioreactor to the seed bioreactor depends uponthe degree to which the cell line is propagated in the first bioreactor,but is typically from 3:1 to 10:1, e.g., in the range of (6-8):1.According to a preferred embodiment, the cultivation vessel is acontinuous stirred tank bioreactor that allows control of temperature,aeration, pH and other controlled conditions and which is equipped withappropriate inlets for introducing the cells, sterile oxygen, variousmedia for cultivation and outlets for removing cells and media and meansfor agitating the culture medium in the bioreactor.

According to the present invention, “serum-free medium” (SFM) meant acell culture medium ready to use, that is to say that it does notrequired animal serum addition allowing cells survival and cell growth.This medium is not necessary chemically defined, and may containedhydrolyzates of various origin, from plant or yeast for instance.Preferably, said SFM are “non animal origin” qualified, that is to saythat it does not contain components of animal or human origin (FAOstatus: “free of animal origin”). In SFM, the native serum proteins arereplaced by recombinant proteins. Alternatively SFM medium according tothe invention does not contain protein (PF medium: “protein freemedium”) and/or are chemically defined (CDM medium: “chemically definedmedium”). SFM media present several advantages: (i) the first of allbeing the regulatory compliance of such media (indeed there is no riskof contamination by adventitious agents such as BSE, viruses): (ii) theoptimization of the purification process; (iii) the betterreproducibility in the process because of the better defined medium.Examples of commercially available SFM media are: VP SFM (InVitrogen Ref11681-020, catalogue 2003), Opti Pro (InVitrogen Ref 12309-019,catalogue 2003), Episerf (InVitrogen Ref 10732-022, catalogue 2003), Pro293 S-CDM (Cambrex ref 127650, catalogue 2003), LC17 (Cambrex RefBESP302Q), Pro CHO 5-CDM (Cambrex ref 12-766Q, catalogue 2003), HyQSFM4CHO (Hyclone Ref SH30515-02), HyQ SFM4CHO-Utility (Hyclone RefSH30516.02), HyQ PF293 (Hyclone ref SH30356.02), HyQ PF Vero (HycloneRef SH30352.02), Excell 293 medium (SAFC Biosciences ref 14570-1000M),Excell 325 PF CHO Protein free medium (SAFC Biosciences ref14335-1000M), Excell VPRO medium (SAFC Biosciences ref 14560-1000M),Excell 302 serum free medium (SAFC Biosciences ref 14312-1000M), Excell65319, Excell 65421, Excell 65625, Excell 65626, Excell 65627, Excell65628, Excell 65629 (JRH Biosciences), Excell MDCK SFM (SAFC-BiosciencesRef. 14581C), Excell MDCK Prod (Ref. M3678), Gene Therapy Medium 3(animal component free) (SIGMA-Aldrich, ref. G-9916 or Excell GTM-3)(hereinafter named G9916 medium), HYQ CDM4 HEK-293 (Hyclone Ref.SH30859), HYQ SFM4 HEK-293 (HYCLONE Ref. SH30521), AEM (InVitrogen).According to the first preferred embodiment, the serum-free medium No 1and the serum-free medium No 2 are the same medium. According to asecond preferred embodiment the serum-free medium No 1 and theserum-free medium No 2 have a different composition.

The process of the invention encompasses the removal of the whole or apart of serum-free medium 1, followed by its replacement by serum-freemedium No 2. However, it is more convenient to remove a substantialfraction (e.g., up to about 50%) of the serum-free medium 1 and thenreplenish it with the serum-free medium No 2 while still removing medium1, e.g., through the spinfilter. According to a preferred embodiment,serum-free medium No 2 is directly added to serum-free medium No 1without removal of a part of serum-free medium No 1. Between 0.25 to 10volumes of serum-free medium No 2 is added to 1 volume of serum-freemedium No 1. In a preferred embodiment, between around 0.5 to 8 volumesof serum-free medium No 2 is added to 1 volume of serum-free mediumNo 1. In a more preferred embodiment, between around 3 to 6 volumes ofserum-free medium No 2 is added to 1 volume of serum-free medium No 1.

The serum-free medium No 1 and/or the serum-free medium No 2 may besupplemented with at least one ingredient selected from the groupconsisting of amino-acids, lipids, fatty acids, cholesterol, vitamins,carbohydrates, protein hydrolyzates of non-animal origin, and a mixturethereof.

Alternatively, the process of replicating a virus of the invention is afed-batch process that comprises the additional step of feeding thecells with at least one ingredient selected from the group consisting ofamino-acids, lipids, vitamins, carbohydrates, protein hydrolyzates ofnon-animal origin, surfactant and a mixture thereof. According to afirst preferred embodiment, the feeding occurs during steps a) to d) ofthe process of the invention of replicating a virus, alternatively onlyduring the steps b) to d), or alternatively only during the steps d).The feeding may occur either on a daily basis or on a continuous basis.When the feeding is discontinuous, the feeding may occur one time perday, more than one time per day, or less than one time per day.

The SFM media of the invention comprise a number of ingredients,including amino acids, vitamins, organic and inorganic salts, sources ofcarbohydrate, each ingredient being present in an amount which supportsthe cultivation of a cell in vitro. However, in order to improve cellgrowth or viral productivity, additional ingredients are added to SFMmedia.

The choice of amino-acid(s) to add to the cell culture may be determinedbe an analysis of amino-acids consumption by the cells in the culture;such consumption varies according to cell species. According to apreferred embodiment, the amino-acids added to the medium may beselected from the group consisting of asparagine and glutamine, or amixture thereof. In a more preferred embodiment, glutamine is added forchicken EBx cell culture and the feeding of glutamine is performedduring step a) to d) to maintain the glutamine concentration in themedium between around 0.5 mM to around 5 mM, preferably between around 1mM to around 3 mM, and most preferably around 2 mM. In a preferredembodiment, the feeding of glutamine occur on a continuous basis.Interestingly, duck EBx® cells do not consume much glutamine, becauseduck cells have the ability to synthetize glutamine. Therefore,glutamine may or may not be added for duck EBx cell culture.

According to a preferred embodiment, the carbohydrates added to themedium are selected from the group consisting of D-glucose, D-sucroseand D-galactose or a mixture thereof. According to a more preferredembodiment, the carbohydrate added is D-glucose. The feeding ofD-glucose is performed during step a) to d), more preferably between b)to d) to maintain the D-glucose concentration in the medium betweenaround 0.5 g/l to 25 g/l of D-glucose, preferably between around 1 g/lto 10 g/l of D-glucose, preferably around 2 to 3 g/l of D-glucose. In apreferred embodiment, the feeding of D-glucose occur on a continuousbasis.

According to a preferred embodiment, the lipids are selected from thegroup consisting of cholesterol, steroids, and fatty acids such aspalmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, linolenic acid, and their derivatives, or a mixture thereof. Morepreferably the fatty acids are from SIGMA-ALDRICH (Ref. F7050) andaround 0.35 μl/ml of fatty acids solution is added to the culturemedium.

The medium may contain auxiliary substances, such as buffer substanceslike sodium bicarbonate, oxidation stabilizers, stabilizers tocounteract mechanical stress, or protease inhibitors. If required, anon-ionic surfactant, such as polypropylene glycol (PLURONIC F-61,PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108) can beadded to the medium as a de-foaming agent. These agents are generallyused to protect cells from the negative effects of aeration since,without an addition of a surfactant, the ascending and bursting airbubbles can lead to damage of those cells that are located on thesurface of these air bubbles (“sparging”). The quantity of nonionicsurfactant is preferably between about 0.05 and about 10 g/L, typicallybetween about 0.1 and about 5 g/L According to another embodiment of theinvention, the concentration of surfactant in cell culture medium may bemodified to adapt (i.e increase or decrease) the size of the cellclumps.

According to an embodiment of the process of replicating a virus of theinvention, the addition of serum-free medium No 2 to the cell culture,is performed after infection step b), preferably between around 0.5 to 4hour after step b), and more preferably around 1 hour after step b).According to another embodiment of the invention, the addition ofserum-free medium No 2 to the cell culture, is performed beforeinfection step b), preferably between around 0.5 to 4 hour after stepb), and more preferably around 1 hour before step b). According toanother embodiment of the invention, the addition of serum-free mediumNo 2 to the cell culture, is performed simultaneously to infection stepb. The viral infection of step b) is carried out at an m.o.i(multiplicity of infection) of about 10 to 10⁻⁸, preferably 10⁻¹ to10⁻⁶, more preferably about 10⁻² to 10⁻⁵, and more preferably about10⁻⁴. The man skilled in the art will determine the optimal m.o.iaccording to the virus type. In step c), the infected cells arepreferably cultured during at least 24 h, at least 48 h, at least 72 h,at least 96 h, at least 120 h, at least 144 h. When the virus is apoxvirus, the infected cells are cultured at least 144 h.

In the process of the invention, the cell culture of step a) is carriedout by batch culture, repeated batch culture, fed-batch culture orperfusion culture. More preferably, the cell culture of step a) isperformed by fed-batch culture. The infection in step b) is performedwhen the cell density is at least around 4 million, preferably 6 millioncells/ml, more preferably 8 million cells/ml in batch or led-batchprocess. When a perfusion process is used, the infection in step b) isperformed when the cell density is of at least at least 8 millioncells/ml, preferably around 9 to 10 million cells/ml, or even higher.

The pH of the serum-free culture medium in steps a), b), c) and d) ispreferably monitored by the bioreactor. The pH shall be in a range from6.5 to 7.8, preferably around 6.8 to 7.5, and more preferably around7.2.

In the process of the invention, step d) lasts for 1 to 10 days beforethe harvest. According to a preferred embodiment, step d) lasts for 2 to5 days before the harvest. The time of harvest (step e) is definedaccording to the cell density in the cultivation vessel. The inventorhave now found that the optimal time for harvest the viruses is two daysalter the density of viable cells have reached its optimal level andhave started to decrease because of viral infection.

The cell culture is performed at a temperature comprises between 32° C.to 39° C. depending of the virus type. For influenza virus and poxvirusproduction, cell culture infection is preferably performed at 33° C.

EBx® cells have the ability to grow in suspension culture with cellsclumped in loose aggregates of few cells, up to more than hundred(s) ofcells. Without to be bind by a theory, the size of the clumps may varyaccording to the composition of cell culture medium. For example,presence of surfactant such as polypropylene glycol (PLURONIC F-61,PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108), thestirring, the concentration of divalent ions, such as Mg2+ and Ca2+, mayhave an effect on the clumps size. The inventor has now found that theviral yield may be increased by allowing the EBx® cells of the inventionto aggregate to each others to form clumps during at least step a) ofthe process. During the scaling-up from the master and working cell bankvial through the various sizes of T-flasks or roller-bottles tobioreactors, the suspension cells are generally passaged to a largervessel, either by dilution into fresh medium or by centrifugationfollowed by a re-suspension of cell pellet into a fresh medium. Theinventor has found that during the cells passages, it is recommended tokeep large cell clumps into the culture. To do so, it is better not todisrupt cells clumps in order to improve the replication of virus inEBx® cells. For example, during the initial phases of culture of step a)in T-flasks or roller-bottles, it is recommended to dilute the cellculture to passage the cells into larger vessel(s), and it is notrecommended to centrifuge, nor to disrupt the cells clumps by pipettingor stirring. However, too large clumps may be suboptimal for a highviral production. Consequently, the man skilled in the art will definewhether a partial disruption of the clumps, by pipetting or stirring,during initial cell passages of step a) may improve viral yield.According to a preferred embodiment, poxviruses, and preferably MVA,ALVAC and Fowlpox viruses are obtained by a process of the inventionthat include the step a) of proliferating clumped EBx® in looseaggregates of few cells, up to more than at least one hundred of cells,at least two hundred of cells, at least five hundred of cells, at leastthousand(s) of cells.

The inventors have found that size of EBx® cells clumps, preferably duckEBx® cells clumps, may be dependent of Mg2+ and/or Ca2+ ionsconcentration in anchorage-independent cell culture medium. Since toolarge clumps may be suboptimal for a high viral production, the size ofclumps may be monitored by adjusting Mg2+ and Ca2+ concentration in cellculture medium. For duck EBx® cells, the cell culture medium preferablycontain Mg2+ concentration comprises between 0.5 mM and 2.5 mM,preferably around 1.6 mM, and Ca2+ concentration comprises between 0.01mM and 0.5 mM, preferably around 0.1 mM.

The invention also relate to the virus obtainable by a process of theinvention. The instant invention also relates to the vaccine containingthe virus of the invention. The process of manufacturing a viral vaccinecomprises the process of replicating a virus according to the inventionwherein the step e) of virus harvest is comprising at least one stepselected among filtering, concentrating, freezing and stabilizing byaddition of stabilizing agent. The virus harvest is performed accordingto technologies well-known to the man skilled in the art. According to apreferred embodiment, the step of harvesting said virus comprisescollecting cell culture supernatant obtained from centrifugation of cellculture, then filtering, concentrating, freezing and stabilizing viruspreparation by addition of stabilizing agent. For example, for influenzavirus see Furminger, In Nicholson, Webster and Hay (Eds) Textbook ofinfluenza, chapter 24 pp 324-332.

The process of manufacturing a viral vaccine according to the inventionmay also comprise the additional step of inactivation of harvestedvirus. Inactivation is preferably performed by treatment withformaldehyde, beta-propiolactone, ether, ether and detergent (i.e suchas Tween 80™), cetyl-trimethyl ammonium bromide (CTAB) and Triton N102,sodium deoxycholate and tri(N-butyl)phosphate.

According to another embodiment, the invention also relates to a processof preparation of viral antigenic proteins from the virus obtainable bya process of the invention, said process comprises the additional stepsof:

-   -   a) optionally, incubating cell culture supernatant comprising        whole virus with a desoxyribonucleic acid restriction enzyme,        preferably DNAses (see EC3.1.21 and EC3.1.22 classification) and        nucleases (see EC3.1.30 and EC3.1.31 classification).        Preferably, DNA digestion enzyme is benzonase (Benzon nuclease)        or DNase I;    -   b) adjunction of cationic detergent. Examples of cationic        detergent are; without limitation: cetyl-trimethyl ammonium salt        such as CTAB, myristyl-trimethyl ammonium salt, lipofectine,        DOTMA and Tween™;    -   c) isolation of antigenic proteins. This latter step may be        realized by centrifugation or ultrafiltration.

The virus in the vaccine may be present either as intact virusparticles, or as disintegrated virus particles. According to anembodiment, the vaccine is a killed or inactivated vaccine. According toanother embodiment, the vaccine is a live attenuated vaccine whereinsaid vaccines mainly comprises EBx cell culture supernatant obtainableby the process of the invention, preferably without serum, optionallyfiltered and/or concentrated and comprising said virus. According to athird embodiment, the vaccine is comprising viral antigenic proteinsobtainable from a virus prepared according to the process of theinvention.

The invention also pertain to provide a vaccine comprising an infectedcell line EBx®, preferably duck or ev-0 chicken EBx®, obtainable by theprocess of the invention, and wherein infected cell line EBx®,preferably preferably duck or ev-0 chicken EBx®, are harvested in stepd).

The vaccine of the invention may comprise the virus of the invention incombination with pharmaceutically acceptable substances which increasethe immune response. Non limitating examples of substances whichincrease the immune response comprises incomplete Freund adjuvant,saponine, aluminium hydroxide salts, lysolecithin, plutonic polyols,polyanions, peptides, bacilli Calmette-Guerin (BCG) and corynebacteriumparvum. Example of synthetic adjuvant is QS-21. In addition,immuno-stimulating proteins (interleukins Il1, Il2, IL3, IL4, IL12,IL13, granulocyte-macrophage-colony-stimulating factor, . . . ) may beused to enhance the vaccine immune response.

The vaccine of the invention is preferably a liquid formulation, afrozen preparation, a dehydrated and frozen preparation, optionallyadapted to intra-nasal route of administration.

The vaccine of the invention is use for the prophylactic and/ortherapeutic treatment of a human or an animal infected by a viruspreviously listed. Preferably, the viral vaccine of the invention ispreferably use for the prophylactic and/or therapeutic treatment of ahuman infected by a virus selected among smallpox, influenza, measles,mumps, rubella viruses, RSV. Alternatively, the vaccine of the inventionis preferably use for the prophylactic and/or therapeutic treatment of aanimal infected by a virus selected among influenza, Newcastle DiseaseVirus, Egg Drop Syndrome Virus, Infectious Bursal Disease, InfectiousBronchitis Virus, Canine Distemper virus, Chicken Anemia Virus. Therecombinant viral vaccine of the invention may also be used for theprophylactic and/or therapeutic treatment of chronic diseases such ascancer and infectious diseases such as AIDS.

The EBx® cell lines of the invention are useful to generate and producere-assorted virus. The virus with a segmented genome, such as influenzavirus may be re-assorted. When infecting simultaneously EBx® cells ofthe invention with at least two different strains of influenza virus, amix of segmented genome from two different strains is present in thesame host cell. During virus assembly, all combination of genomicsegments can theoretically be generated. Specific reassorted virus maythus be isolated by selecting or eliminating, with an antibody forexample, virus with a desired traits (See Kilnourne E. D in Plotkin S Aand Mortimer E. A. Eds, Vaccines 1994). The EBx® cell lines of theinvention are also useful to generate and produce influenza virus byreverse genetics (See Enami, Proc. Natl. Acad. Sci. USA, 87:3802-3805(1990); Enami et Palese, J. Virol. 65:2511-2513 (1991); Luytjes, Cell59:1107-1113 (1989)).

The present invention also relates to the use of EBx® cell lines of theinvention as a cell substrate to perform virus titration. EBx® cellswill efficiently in replace current cell system, such as embryonatedeggs, CEFs, DF1 cells and others, used to determine the titer of a viralsolution. Preferable the viral titration is performed by TCID50 method(Reed L, Muench H, 1938. A simple method of estimating fifty percentendpoints. Am. J. Hyg. 27, 493-97).

The present invention also relates to the use of EBx® cell lines of theinvention as a cell substrate to perform sanitary testing.

The invention also relates to the diagnostic composition containingviruses of the invention or constituents thereof.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims. For the remainder of the description, referencewill be made to the legend to the figures below.

FIGURES

FIG. 1: Anchorage-Independent chicken EBx cells

FIG. 1A: Anchorage-Independent chicken Valo EBv13 cells In serumfree-medium. EBv13 cells were cultured at 37° C. in suspensionserum-free medium Excell 65319 (SAFC). EBv13 cells have an homogeneoussize and grow in loose clumps into culture. The population doubling timeis about 16-18 Hours and the cell density reached in agitated flaskvessels were about 4-5 millions cells/ml.

FIG. 1B: Anchorage-independent chicken EB Line 0 cells in serumfree-medium

EB Line 0 cells were cultured at 39° C. in suspension serum-free mediumExcell 66444 (SAFC). EB Line 0 cells have an homogeneous size and growin loose clumps.

FIG. 2: Chicken Valo EBv13 cells express high level of Telomerase

EBv13 cells at passage p193 do express high level of telomerase in thesame order of magnitude that chicken EB14-O74 cells (see WO03/076601) atpassage p164 (Master cell Bank: MCB) or at passage p184 (Workin Cellbank: WCB). Murine embryonic stem cells (mES) were used as a positivecontrol and mouse fibroblast (FED) were used as a negative control.

FIGS. 3A and 3B: Susceptibility of Chicken Valo EBv13 to poxvirus

EBv13 (passage 188) were seeded at 0.4×106 cells/ml in 100 mL F175flasks either in 40 ml of SFM Excell Medium 65319 or G9916 SFM Medium(SAFC) supplemented with 4 mM Glutamine. The cell growth and infectionwith MVA-GFP (MOI 10⁻² TCID50/cell) were performed at 37°. One hour postinfection, 60 ml of fresh medium were added.

FIG. 3A: cell density kinetics in SFM Excell Medium 65319 or G9916 SFMMedium (SAFC).

FIG. 3B: MVA productivity expressed in TCID50/ml in SFM Excell Medium65319 or G9916 SFM Medium (SAFC).

FIGS. 4A. 4B and 4C: Transmission Electronic Microscopy analysis of duckEBx cells

FIG. 4A: Micrograph of anchorage-independent duck EBx cells.

FIG. 4B: Transmission Electronic Microscopy analysis of dEBx cells wereperformed by Dr. A Rivoire (Lyon, France). Duck EBx cells display atypical embryonic stem cells morphology (i.e high nucleo-cytoplasmicratio) that resemble the phenotype of murine embryonic stem cells andVIVALIS EB14 cells described in WO2006/108846. Duck EBx cells are smallround cells with a large nucleus and nucleolus, with short pseudopodiaextending from the plasma membrane. They are highly metabolic activewith a ribosome and mitochondria rich cytoplasm.

FIG. 4C: Micrograph of duck EBx cells able to grow in suspension,isolated during feeder deprivation, and adapted to grow withoutadditives and serum.

FIGS. 5A and 5B: Telomerase expression In duck EBx cell lines

Telomerase expression during different stages of establishment of duckEBx cells was investigated by using Roche telomerase detection kit(Telomerase OCR ELISA).

FIG. 5A: Telomerase is found to be highly expressed in differentadherent duck EBx cell lines just like in chicken EBv13 cells. Duckepithelial cells used as a negative control do not express telomerase.

FIG. 5B: During the process of establishment of suspension duck EBxcells, high level of telomerase expression is maintained. High level oftelomerase were investigated in duck EBx cells during feeder deprivation(with or without feeder cells), during the process of adapting duck EB26cells to suspension and after serum deprivation of dEB24 et dEB26.

Duck EBx cells, such as EB24 and EB26, express high level of telomerasejust like chicken EB14 cells. Duck EB66 also express high level oftelomerase (Data not shown).

FIGS. 6A and 6B: Duck EBx® cells display no endogenous reversetranscriptase activity

FIG. 6A: Endogenous reverse transcriptase expression was investigated bydirect F-PERT analysis (Lovatt et al., 1999, J. Virol. Methods,82:185-200) in Clean Cells (FRANCE). Duck EBx® cell lines, EB26 andEB5-1, display no endogenous Reverse Transcriptase (RT) activity. Highlevel of RT activity were detected in chicken EB14 and EBv13 cellsculture (at different passages) as well as, to a lesser extend, inchicken embryonic libroblast (CEF) derived from Specific Pathogen Free(SPF) chicken strain. CEM cells, which are RTase negative, were used asa negative control to set the detection limit of the assay.

FIG. 6B: Presence of endogenous retroviral particles, either replicative(i.e replication competent) or non-replicative, in the cell culturesupernatant of duck and chicken EBx cells were investigated by an ELISAassay detecting the avian leukosis major capsid antigen P27. Duck EBxcell lines, EB26 and EB5-1, as well as chicken EBv13 do not secrete ALVp27 antigen. In the opposite, chicken EB14 cells do express ALV P27antigen.

FIGS. 7A and 7B: Duck EBx cells do not secrete replicative avianleucosis virus (ALV)

Co-cultivation assay of duck EBx cells with quail QT6 cell line, knownto be sensitive to endogenous and exogenous ALVs, were performed inBioreliance (UK) to detect the presence of endogenous replicative duckviruses.

FIG. 7A: described the principle of QT6 co-culture.

FIG. 7B: The presence of replicative virus is detected by an ELISA assaydetecting the avian leukosis major capsid antigen P27.

The assay demonstrates that none of duck EBx® cells tested (dEB26 anddEB51) secrete replicative ALV. RAV-1 virus, which is known to replicatein QT6, were used as a positive control.

FIG. 8: Cell surface expression of receptors SAo2-3 and SAa2-6 in duckEBx and chicken EB14 cell lines

Cells are incubated with digoxygenin labelled lectins: Sambuca nigraagglutinin lectin specifically binds to Sia2-6Gal, while Maackiaamurensis agglutinin lectin specifically binds to Sia2-3Gal. Lectinsthat bind to cells are revealed with anti-digoxygenin antibodyFITC-labelled according to well-known techniques by the man skilled inthe art. FITC-labelled cells are numbered with a fluorescent cell sorter(FACS). SAa2-3 and SAa2-6 molecules are been described to be thereceptors for the avian and human influenza viruses, respectively.Almost all duck EBx cells highly express cell surface receptors SAa2-3and SAa2-6.

FIGS. 9A and 9B: MVA-GFP virus propagation in infected duck EBx cells

FIG. 9A: Duck EBx® were allowed to form small clumps in T175 stirredtank flasks during cell proliferation in a cell growth SFM medium.Clumps were then infected with 10⁻² TCID₅/cell of MVA-GFP virus and themixture was diluted in production SFM media. During a 6 days viruspropagation period at 37° C., pictures of UV-exposed infected cells weretaken daily. The pick of MVA infection was reached at day 4post-infection (pi). At day 6 μl, the infected cells start to die.

FIG. 9B: MVA-GFP virus titration propagated in duck EBx® cells in a 3 Lfed-batch bioreactor. (Left Panel) Duck EBx-derived biomass was allowedto accumulate during cell proliferation phase in Excell growth medium(SAFC). At day 4, cell density reached 4 million cells/ml. Cells werethen infected with 10⁻¹ TCID₅₀/cell of MVA-GFP virus and the mixture wasdiluted in 1.5 L Excell medium. During a 6 days virus propagation periodat 37° C., samples were collected daily and TCID₅₀ titration (RightPanel) was performed at the end of the kinetic. A yield of 8.5 logTCID50/ml were reached at 4 p.i. corresponding to a yield of 205TCID₅₀/Cell.

FIGS. 10A and 10B: Influence of Calcium and Magnesium concentration InSFM medium on the size of EBx® cells clumps

FIG. 10A: Chicken EBv13 cells were first cultured in the SFM medium fromSAFC Biosciences that comprise a high concentration of calcium (Ca2+)(Approx. 0.79 mM) and magnesium (Mg2+) ions; in this medium, cellsproduce large aggregates in culture.

Three days alter having changed the cell culture medium with the sameSFM medium that comprises a lower concentration of Ca2+(0.03 mM final)and Mg2+(1.6 mM final), the cells form smaller aggregates.

FIG. 10B: Duck EB24, EB26 and EB66 cells were first cultured in the SFMmedium from SAFC Biosciences that comprise a high concentration ofcalcium (Ca2+) (Approx. 0.79 mM) and magnesium (Mg2+) ions; in thismedium, cells produce large aggregates in culture.

Three days after having changed the cell culture medium with the sameSFM medium that comprises a lower concentration of Ca2+(0.03 mM final)and Mg2+(1.6 mM final), the cells form smaller aggregates.

FIGS. 11A and 11B: Production of Influenza virus strains A In duck EBxcells In 3 L-bioreactors

Duck EBx® biomass was allowed to accumulate at 37° C. during cellproliferation phase in a cell growth medium. Cells were then infectedwith 10⁻⁴ TCID₅₀/cell of A/H1N1/Beijing/262/95 or A/H3N2/NewYork/55/2004 influenza virus, the mixture was diluted in 1.5 L Excellproduction medium supplemented with 0.75 USP/mL of trypsin andtemperature was lowered to 33° C. During a 14 days virus propagationperiod, samples were collected daily and stored at −80° C.

FIG. 11A: Growth kinetic of duck EBx cells infected withA/H1N1/Beijing/262/95 influenza virus strain

Left panel: Cell density (rhombus, ×10⁶ cells.ml⁻¹) and viral titer inlogTCID₅₀/ml.

Right panel: Total number of cells (square), viability (black circles,%) and haemagglutinin concentration in ug/ml (red circles, %).

The viral yield reached 20 ug of Hemagglutinin per ml of culturesupernatant.

FIG. 11B: Growth kinetic of duck EBx cells infected with A/H3N2/NewYork/55/2004 influenza virus strain

Left panel: Cell density (rhombus, ×10⁶ cells.ml⁻¹)

Right panel: Total number of cells (square), viability (black circles,%) and haemagglutinin concentration in ug/ml (red circles, %).

The viral yield reached 30 ug of Hemagglutinin per ml of culturesupernatant.

FIG. 12: Production of influenza virus strain B In duck EBx® cells

Duck EBx® biomass was allowed to accumulate at 37° C. during cellproliferation phase in a cell growth medium. Cells were then infectedwith 10⁻³ TCID₅₀/cell of B/Jiangsu/10/2003 influenza virus, the mixturewas diluted in 1.5 L Excell production medium supplemented with 0.75USP/mL of trypsin and temperature was lowered to 33° C. During a 14 daysvirus propagation period, samples were collected daily and stored at−80° C.

Left panel: Cell density (rhombus, ×10⁶ cells.ml⁻¹)

Right panel: Total number of cells (square), viability (black circles,%) and haemagglutinin concentration in ug/ml (red circles, %).

The viral yield reached 25 ug of Hemagglutinin per ml of culturesupernatant.

FIG. 13: Analysis of NDV productivity and viral protein expression insuspension duck EB66 cells (MOI 10³, 0.75 USP/mL trypsin)

Duck and chicken EBx cells are sensitive to and replicate NDV La Sotastrain. Titers (in TCID50/ml) of NDV produced in duck EB66 cellsincrease from day 0 to day 2 μl to reach an average of 10^(6.83)_(TCID50)/mL (FIG. 13 Left Panel).

Western blot analysis (FIG. 13 right Panel) showed NDV viral proteins(HN, Fo/F, NP & M) expression. The viral proteins composition of NDVvirus produced in duck EB66 cells are similar to the one obtained withNDV virus produced in chicken EB14 cells. In addition, the kinetic ofrelease for viruses produced in chicken and Duck EBx cells are similar.

FIG. 14: Analysis of recombinant Measles virus replication In suspensionduck EB66 cells (MOI 10⁻¹ or 10⁻²) In tissue-culture flasks In serumfree medium. Duck EB66 cells are at least as sensitive as VERO cells toinfection by Measles Virus. Titers (in TCID50/ml) of recombinant Measlesvirus expressing Green Fluorescent Protein (GFP) produced in duck EB66cells reach 10⁷ TCID50/mL at day 6 post-infection.

FIGS. 15A and 15B: SSEA-1, EMA-1 & Telomerase expression in duck EB66cells Telomerase expression at different passages of duck EB66 culturedin roller bottles was investigated by using Roche telomerase detectionkit (Telomerase OCR ELISA). SSEA-1 and EMA-1 at different passages ofduck EB66 cultured in roller bottles was investigated by FACS analysis.

FIG. 15A: Telomerase is found to be highly expressed in suspension duckEB66 cell line at different passages (138, 144, 147, 150, 154).

FIG. 15B: SSEA-1 and EMA-1 cell surface markers was found to be highlyexpressed in suspension duck EB66 cell line at different passages (138,144, 147, 150, 154).

FIG. 16: Karyotype analysis of duck EB66 cells

Duck EB66 cells karyotype was performed by Pr. Franck, ENVL, Lyon. EB66cells are diploid cells.

EXAMPLES Example 1 Chicken EBv13 Cell Line from SPF Chicken Strain VALO1.1—Raw Material Eggs

Specific Pathogen Free (SPF) strain called Valo. The valo strain is awhite Leghorn strain produced and delivered by Lohmann from Germany.Those SPF chicken eggs, supplied with a certificate of analysis, aretested for: CAV, Avian adenoviruses (group 1, serotypes 1-12 and group3), EDS, Avian Encephalomyelitis Virus, Avian Leukosis Viruses/RSV(including Serotype ALV-J), Avian Nephritis Virus, Avian Reoviruses,Fowlpox Virus, Infectious Bronchitis Virus, Infectious Bursitis Virus(IBDV), Infectious Laryngo Tracheitis Virus, Influenzavirus Typ A,Marek's Disease Virus, Mycoplasmosis (Mg+Ms), Mycobacterium avium,Newcastle Disease Virus, Reticuloendotheliosis Virus, Salmonellapullorum, Other Salmonella Infections, Avian Rhinotracheitis Virus(ART), Hemophilus paragallinarum. Valo chicken eggs were only submittedto a disinfection with the decontaminant to avoid any risk ofcontamination linked to the manipulation of eggs during the transport.

Feeder Cells

In the first step of the process of establishment of EBv13, cells frommurine origin (STO cells) were used as feeder layer to maintain thepluripotency of chicken stem cells. Those feeder cells are mitoticallyinactivated by gamma irradiation (45 to 55 Grays) before seeding onplastic. This dose of irradiation is a sub-lethal dose that induces adefinitive arrest of the cell cycle but still permits the production ofgrowth factors and extracellular matrix, necessary for the promotion ofthe cell growth of non differentiated cells.

The STO cell line was derived by A. Bernstein, Ontario Cancer Institute,Toronto, Canada from a continuous line of SIM (Sandos Inbred Mice) mouseembryonic fibroblasts and it was supplied by the American Type CultureCollection (ATCC) (STO Product number: CRL-1503, Batch number 1198713).Fresh feeder layers were prepared twice a week, in general on Monday andThursday. Exponentially cells were dissociated and counted. A part ofcells were seeded for maintenance of viable cultures and another partwas irradiated. For irradiation, we prepared a cell suspension at 10×10⁶cells/mL in tubes. Cells were exposed to a 45 to 55 grey dose and wereseeded on plastic. After seeding, dishes or plates coated withinactivated feeder cells were used during a maximum of 5 days

Medium

-   -   DMEM-HamF12 (Cambrex, Cat no BE04-687)    -   Optipro medium (Invitrogen, Cat no 12309)    -   EX-CELL™ 65195. 60947 and 65319 (SAFC, customized medium)

Additives

-   -   Glutamine (Cambrex, Cat no BE17-605E)    -   Pencillin/streptomycin (Cambrex, Cat no BE17-602E))    -   Non essential Amino Acids (Cambrex, Cat no BE13-114E)    -   Sodium pyruvate (Cambrex, Cat no BE13-115)    -   Vitamines (Cambrex, Cat no 13-607C)    -   Beta Mercapto Ethanol (Sigma, Cat no M7522)

Buffer and Fixators

-   -   PBS 1× (Cambrex, Cat no BE 17-516F)    -   Paraformaidehyde 4% (Sigma, Cat no P6148)    -   KCl 5.6% (Sigma, Cat no P9333)    -   Methanol/Acetic acid (3/1): Methanol (Merck, Cat no K34497209;        Acetic acid Sigma Cat no A6283) Colcemid. Karyomax (Gibco, Cat        no 15212-046)

Cryoprotective Agent

-   -   Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650)

Factors

Two different recombinant factors were used:

-   -   Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech        Inc, Cat no 450-13)    -   Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc,        Cat no 100-11)        The two factors were produced in E. Coli bacteria.

Fetal Bovine Serum

Non Irradiated Fetal Bovin Serum (FBS) (JRH, Cat No 12103)

The non irradiated serum used in the program was collected and producedin United States. Animals used for collection were USDA inspected andacceptable for slaughter. It was added in the medium during avian stemcells culture. This batch was not submitted to irradiation to avoid thedestruction of critical proteins or components identified as essentialfor the maintenance of stem cells in culture.

Irradiated Serum (JRH, Cat No 12107)

The irradiated batch used in this program was also collected in UnitedStates. This irradiated batch was added as supplement in the DMEM mediumused for the culture of STO or FED cells (feeder cells). Those cells donot require as stem cells a specific quality of serum for growth andmaintenance in culture. To minimize high concentration of serum in themedium we have adapted the STO cells to grow in presence of 4% of FBSonly.

Dissociating Agents:

Pronase (Roche, Cat No 165 921)

Pronase is a recombinant protease manufactured by Roche Diagnostics,Germany, used for the dissociation of adherent avian stem cells.

Trypsine EDTA (Cambrex, Cat No BE17-161E)

Trypsine is used for the dissociation of STO or FED cells and at latepassages for the dissociation of avian cells adapted to Serum FreeMedium. This enzyme of porcine origin is manufactured asepticallyaccording to cGMP referential conditions by a validated sterilefiltration method and tested according to current E.P. The raw material,irradiated prior to formulation, is tested for porcine parvovirus instrict compliance with 9/CFR 113.53.

Non Enzymatic Cell Dissociation Solution (Sigma, Cat No C5914)

This agent of dissociation is a ready to use formulation used to gentlydetach cells from the growing surface of the culture vessel. The formulacontains no protein, and allows dislodging of cells without use ofenzymes. Cellular proteins are preserved making possible immunochemicalstudies that are dependent upon the recognition of cell surfaceproteins. This enzyme was used to detach cell before FACS analysis ofbiological markers like EMA-1 (Epithelial Membrane Antigen 1) and SSEA1(Stage Specific Embryonic antigen-i).

1.2—Process of Establishment of EBv13 Cell Line

Eggs are opened, the yolk were separated from the albumen during theopening. The embryos were removed from the yolk either directly with theaid of a Pasteur pipette, or with the aid of a small absorbent filterpaper (Whatmann 3M paper), cut out beforehand in the form of aperforated ring with the aid of a punch. The diameter of the perforationwere about 5 mm. These small rings were sterilized using dry heat forabout 30 minutes in an oven. This small paper ring is deposited on thesurface of the yolk and centered on the embryo which is thus surroundedby the paper ring. The latter is then cut out with the aid of smallpairs of scissors and the whole removed is placed in a Petri dish,filled with PBS or with a physiological saline. The embryo thus carriedaway by the ring were cleaned of the excess yolk in the medium and theembryonic disk, thus free of the excess vitellin, is collected with aPasteur pipette.

The chicken Valo embryos were placed in a tube containing physiologicalmedium (1×PBS, Tris Glucose, medium, and the like). The Valo embryoswere then mechanically dissociated and inoculated on a layer of feederSTO cells into complete culture medium at 39° C. The feeder cells wereseeded in flask at around 2.7×10⁴ cell/cm². The complete culture mediumis composed of basal commercial medium DMEM-Ham F12 supplemented with10% fetal call serum, with IGF1 and CNTF at a final concentration of 1ng/ml, and with 1% non-essential amino acids, with 1% of mixture ofvitamins of commercial origin, with sodium pyruvate at a finalconcentration of 1 mM, with beta-mercapto-ethanol at a finalconcentration of 0.2 mM, glutamine at a final concentration of 2.9 mM,with an initial mixture of antibiotics containing penicillin at a finalconcentration of 100 U/ml and streptomycin at a final concentration of100 μg/ml. Rapidly after the first passages of the cells, the mixture ofantibiotics is no longer added to the medium. The expression rapidly isunderstood to mean after the first 3 to 5 passages in general.

When the avian ES cells from chicken Valo embryos is passaged from aculture dish to another, the seeding of culture dishes was performedwith around between 7×10⁴/cm² to 8×10⁴/cm² of avian ES cells in thecomplete culture medium. Preferably, the seeding is made with around7.3×10⁴/cm² (4×10⁶ cells/55 cm² or 4×10⁶ cells/100 mm dish). The aviancells, preferably the avian embryonic cells of step a) are culturedduring several passages in the complete medium. At passage 15, thecomplete medium was depleted in growth factors IGF1 and CNTF. Thedepletion is made directly in one step, from one passage to another. Theembryonic stem cells, preferably the avian embryonic cells are culturedduring several passages in the complete medium without IGF1 and CNTFgrowth factors.

Then depletion of feeder cells were performed after the depletion ofgrowth factors IGF1 and CNTF by a progressive decreasing of feeder cellsconcentration over several passages. Practically, the same concentrationof the feeder cells were used for 2 to 4 passages, then a lowerconcentration of the feeder cells were used for an additional 2 to 4passages, and so on. The flask were originally seeded with around2.7×10⁴ feeder cells/cm², then around 2.2×10⁴ feeder cells/cm², thenaround 1.8×10⁴ feeder cells/cm², then around 1.4×10⁴ feeder cells/cm²,then around 1.1×10⁴ feeder cells/cm², then around 0.9×10⁴ feedercells/cm², then around 0.5×10⁴ feeder cells/cm². Then the flask wereseeded with 6.5×10⁴ avian cells/cm² to 7.5×10⁴ avian cells/cm² andwithout feeder cells. The depletion of feeder cells started at aroundpassage 21 and ended at around passage 65. During the depletion offeeder cells, the chicken Valo ES cells were seeded in culture flask ata lower concentration than in step a), about around 4×10⁴ cell/cm² to5×10⁴ cell/cm². In the hypothesis that Valo ES cells were not in goodshape following a decrease of feeder cells concentration in the flask,then the avian cells are cultured for additional passages with the samefeeder cells concentration before to pursue the feeder cells depletion.

The serum depletion were performed after the growth factor and thefeeder cells depletion. At the beginning of serum depletion, the culturemedium were composed of basal commercial medium DMEM-HamF12 supplementedwith 10% fetal calf serum and with 1% non-essential amino acids, with 1%of mixture of vitamins of commercial origin, with sodium pyruvate at afinal concentration of 1 mM, with beta-mercaptoethanol at a finalconcentration of 0.2 mM, glutamine at a final concentration of 2.9 mM.The chicken Valo cells were adapted to the growth in a serum free mediumculture in a two steps process: first, the chicken Valo cells wererapidly adapted to a culture medium composed of commercial serum freemedium (SFM), preferably ExCell 60947 (SAFC Biosciences) supplementedwith 10% fetal calf serum and with 1% non-essential amino acids, with 1%of mixture of vitamins of commercial origin, with sodium pyruvate at afinal concentration of 1 mM, with beta-mercaptoethanol at a finalconcentration of 0.2 mM, glutamine at a final concentration of 2.9 mM.Once this rapid adaptation to a new medium (DMEM-HamF12 to Excell 60947)was performed, a second step is performed consisting of a slowadaptation to decreasing concentration of animal serum in the SFM mediumwere initiated. Serum depletion was performed by a progressivedecreasing starting from 10% serum, then 7.5%, then 5%, then 2.5%, then1.25%, then 0.75% of serum concentration in SFM cell culture medium tofinally reach 0% serum in SFM cell culture medium. Serum depletionstarted at passage 103 and ended at passage 135.

At the end of the process of deprivation of serum when the remainingconcentration of serum in SFM medium was either 0.75% or 0%, theadaptation of anchorage-dependent EBv13 cells to suspension culturestarted. Among the several attempts performed to isolateanchorage-independent EBv13 isolates, 62.5% of the attempts weresuccessful and allow to get different isolates of suspension EBv13cells. One isolate of EBv13 cells were selected according to thepopulation doubling time (around 18 h), the optimal cell concentrationinto flask culture (around 4 million cell/ml), the cell viability, thecell culture homogeneity (presence and size of cells clumps) and theeasiness to manipulate the cells (FIG. 1).

At the end of serum depletion, anchorage dependent chicken Valo cells,named EBv13 were able to grow in absence of grow factors, in absence offeeder cells, in serum free medium. EBv13 Cells were then adapted togrowth at 37° C., by progressively decreasing cell culture temperatureof 0.5° C./day.

Example 2 Chicken EB Line 0 Cell Line from SPF Chicken Strain ELL-02.1—Raw Material Eggs:

Chicken Specific Pathogen Free (SPF) strain called ELL-0 (East LansingLine 0) was provided by the Avian Disease and Oncology Laboratory(USDA-ARS-MWA, USA). Those SPF chicken eggs, are produced from a flocktested intensively to various poultry pathogens. Disease tested include:Salmonella pullorum, Salmonella gallinarum, mycoplasma gallisepticum,mycoplasma synoviae, Avian Leukosis virus A-D and J, Marek's diseasevirus, Reticuloendotheliosis virus. Avian adenovirus. Infectiousbronchitis, Infectious bursal disease, Avian Influenza, Newcastledisease, Avian encephalomyelitis and Avian Reovirus. Line 0 chicken eggswere only submitted to a desinfection with the decontaminant to avoidany risk of contamination linked to the manipulation of eggs duringtransportation.

Feeder Cells

In the first step of the process of establishment of EB Line 0, cellsfrom murine origin (STO cells) were used as feeder layer to maintain thepluripotency of chicken stem cells. Those feeder cells are mitoticallyinactivated by gamma irradiation (45 to 55 Grays) before seeding onplastic. This dose of irradiation is a sub-lethal dose that induces adefinitive arrest of the cell cycle but still permits the production ofgrowth factors and extracellular matrix, necessary for the promotion ofthe cell growth of non differentiated cells.

The STO cell line was derived by A. Bernstein, Ontario Cancer Institute,Toronto, Canada from a continuous line of SIM (Sandos Inbred Mice) mouseembryonic fibroblasts and it was supplied by the American Type CultureCollection (ATCC) (STO Product number: CRL-1503, Batch number 1198713).Fresh feeder layers were prepared twice a week. Exponentially cells weredissociated and counted. A part of cells were seeded for maintenance ofviable cultures and another part was irradiated. For irradiation, weprepared a cell suspension at 10×10⁶ cells/mL in tubes. Cells wereexposed to a 45 to 55 grey dose and were seeded on plastic. Afterseeding, dishes or plates coated with inactivated feeder cells were usedduring a maximum of 5 days.

Media

-   -   DMEM-HamF12 (Cambrex, Cat no BE04-687)    -   Medium GTM-3 (Sigma, Cat no G9916)    -   Medium EX-CELL™ 66522, 65788 and 66444 (SAFC, customized medium)

Additives

-   -   Glutamine (Cambrex, Cat no BE17-605E)    -   Pencillin/streptomycin (Cambrex, Cat no BE17-602E))    -   Non essential Amino Acids (Cambrex, Cat no BE13-114E)    -   Sodium pyruvate (Cambrex, Cat no BE13-115)    -   Vitamines (Cambrex, Cat no 13-607C)    -   Beta Mercapto Ethanol (Sigma, Cat no M7522)    -   Yeastolate (SAFC, Cat no 58902C)

Buffer and Fixators

-   -   PBS 1× (Cambrex, Cat no BE17-516F)

Cryoprotective Agent

-   -   Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650))

Factors

Six different recombinant factors were used:

-   -   Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech        Inc, Cat no 450-13)    -   Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc,        Cat no 100-11)    -   Recombinant Human Interleukin 6 (IL6) (Peprotech Inc, Cat no        200-06)    -   Recombinant Human soluble Interleukin 6 receptor (slL6r)        (Peprotech Inc, Cat no 200-06 R)    -   Recombinant Human Stem Cell Factor (SCF) (Peprotech Inc, Cat no        300-07)    -   Recombinant Human basic Fibroblast Growth Factor (bFGF)        (Peprotech Inc, Cat no 100-18B)

All those factors, excepted IL6r, are produced in E. Coli bacteria.Soluble IL6r is expressed in transfected HEK293 cells.

Fetal Bovine Serum

Non Irradiated Fetal Bovin Serum (FBS) (SAFC, Cat No 12003)

The non irradiated serum used in the program was collected and producedin Australia. Animals used for collection were USDA inspected andacceptable for slaughter. It was added in the medium during avian stemcells culture. This batch was not submitted to irradiation to avoid thedestruction of critical proteins or components identified as essentialfor the maintenance of stem cells in culture.

Irradiated Serum (JRH, Cat No 12007)

The irradiated batch used in this program was collected in Australia.This irradiated batch was added as supplement in the DMEM medium usedfor the culture of STO or FED cells (feeder cells). Those cells do notrequire as stem cells a specific quality of serum for growth andmaintenance in culture. To minimize high concentration of serum in themedium we have adapted the STO cells to grow in presence of 4% of FBSonly.

Dissociating Agents:

Trypzean ((Sigma, Cat No 73449)

2.2—Process of Establishment of the Line 0 Cell Line

Embryos from 13 eggs from Line 0 chicken were collected according to theprocess described in Example 1.2. Then, the Line 0 embryos were placedin a tube containing PBS 1×. Embryos were then mechanically dissociatedand inoculated on a layer of feeder STO cells into complete culturemedium at 39° C. The feeder cells were seeded in dishes at around2.7×10⁴ cell/cm². The complete culture medium is composed of basalcommercial medium DMEM-Ham F12 supplemented with 10% fetal calf serum,with IGF1, CNTF, bFGF, IL6, IL6r and SCF at a final concentration of 1ng/ml, and with 1% non-essential amino acids, with 1% of mixture ofvitamins of commercial origin, with sodium pyruvate at a finalconcentration of 1 mM, with beta-mercapto-ethanol at a finalconcentration of 0.2 mM, glutamine at a final concentration of 2.9 mM,with yeastolate 1× and with an initial mixture of antibiotics containingpenicillin at a final concentration of 100 U/ml and streptomycin at afinal concentration of 100 μg/ml. After 7 passages, the mixture ofantibiotics is no longer added to the medium.

When the avian ES cells from chicken Line 0 embryos are transferred froma culture dish to another, the seeding of culture dishes was performedwith around between 7×10⁴/cm² to 8×10⁴/cm² of avian ES cells in thecomplete culture medium. Preferably, the seeding is made with around7.3×10⁴/cm² (4×10⁶ cells/55 cm² or 4×10⁶ cells/100 mm dish). The aviancells, preferably the avian embryonic cells of step a) are culturedduring several passages in the complete medium supplemented with 10 or15% of FBS. At passage 7, the complete medium was depleted in growthfactors bFGF, IL6, IL6r and SCF. The depletion was made directly in onestep, from one passage to another. The embryonic stem cells, preferablythe avian embryonic cells, were cultured during several passages in thecomplete medium without those 4 growth factors. At passage 12, the 2last factors IGF1 and CNTF were removed from the medium and cells wereamplified without factor.

To promote cell growth 3 base medium were used successively: DMEM HamF12 from passage 1 to passage 18, Exell GTM-3 from passage 18 to passage26 and a mixture of Excell 66788 and Excell 66522 after passage 26.

After passage 30, depletion of feeder cells was performed by aprogressive decreasing of feeder cells concentration over severalpassages following the step by step process previously described. Duringthis phase of feeder deprivation, some cells able to grow in suspensionwere isolated using Excell 66444 as growth medium and serum deprivationwas initiated (FIG. 1B).

Example 3 Duck EBx Cell Line EB66 3.1—Raw Material Duck Eggs

Duck eggs from Peking strains GL30 were obtained from GRIMAUD FRERESSELECTION (La Corbiére, Roussay France). The parent ducks werevaccinated against Escherichia Coli (Autogenous vaccine Coli 01 & 02),Pasteurella multocida (Landavax), Duck viral hepatitis (Hepatovax),Erysipelothrix rhusiopathiae (Ruvax), Avian metapneumovirus (Nemovac),Salmonella typhimurium & Enteridis (Autogenous vaccine), Riemerellaantipestifer (Autovaccine Riemerella), Avian metapneumovirus (NobilisRTV inactive) and Erysipelothrix rhusiopathiae (Ruvax). After receipt,fertilized Peking duck eggs were submitted to a disinfection in anhypochloryde bath followed by a decontamination with Fermacidal (Thermo)to avoid any risk of contamination linked to dusts attached on theshell.

Feeder Cells

In the first step of the process, cells from murine origin (STO cells)were used as feeder layer to maintain the pluripotency of duck stemcells. Those feeder cells are mitotically inactivated by gammairradiation (45 to 55 Grays) before seeding on plastic. This dose ofirradiation is a sub-lethal dose that induces a definitive arrest of thecell cycle but still permits the production of growth factors andextracellular matrix, necessary for the promotion of the cell growth ofnon differentiated cells. The STO cell line was derived by A. Bernstein,Ontario Cancer Institute, Toronto, Canada from a continuous line of SIM(Sandos Inbred Mice) mouse embryonic libroblasts and it was supplied bythe American Type Culture Collection (ATCC) (STO Product number:CRL-1503, Batch number 1198713). Fresh feeder layers were prepared twicea week. Exponentially cells were dissociated and counted. A part ofcells were seeded for maintenance of viable cultures and another partwas irradiated. For irradiation, we prepared a cell suspension at 10×10⁶cells/mL in tubes. Cells were exposed to a 45 to 55 grey dose and wereseeded on plastic. After seeding, dishes or plates coated withinactivated feeder cells were used during a maximum of 5 days.

Medium

-   -   Medium EX-CELL™ 65788, 65319, 63066 and 66444 (SAFC, customized        medium)    -   Medium GTM-3 (Sigma, Cat no G9916)    -   DMEM-HamF12 (Cambrex, Cat no BE04-687)    -   DMEM (Cambrex, Cat no BE 12-614F)

Additives

-   -   Glutamine (Cambrex, Cat no BE17-605E)    -   Pencillin/streptomycin (Cambrex, Cat no BE17-602E))    -   Non essential Amino Acids (Cambrex, Cat no BE13-114E)    -   Sodium pyruvate (Cambrex, Cat no BE13-115)    -   Vitamines (Cambrex, Cat no 13-607C)    -   Beta Mercapto Ethanol (Sigma, Cat no M7522)    -   Yeastolate (SAFC, Cat no 58902C)

Buffer and Fixators

-   -   PBS 1× (Cambrex, Cat no BE 17-516F)

Cryoprotective Agent

-   -   Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650)

Factors

Two different recombinant factors were used:

-   -   Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech        Inc, Cat no 450-13)    -   Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc.        Cat no 100-11)

Those 2 factors are produced in E. Coli bacteria.

Fetal Bovine Serum Non Irradiated Fetal Bovin Serum (FBS) (JRH, Cat No12003)

The non irradiated serum used in the program was collected and producedin Australia. Animals used for collection were USDA inspected andacceptable for slaughter. It was added in the medium during avian stemcells culture. This batch was not submitted to irradiation to avoid thedestruction of critical proteins or components identified as essentialfor the maintenance of stem cells in culture.

Irradiated Serum (JRH, Cat No 12107)

The irradiated batch used in this program was collected in UnitedStates. This irradiated batch was added as supplement in the DMEM mediumused for the culture of STO cells (feeder cells). Those cells do notrequire as stem cells a specific quality of serum for growth andmaintenance in culture. To minimize high concentration of serum in themedium we have adapted the STO cells to grow in presence of 4% of FBSonly.

Dissociating Agents:

Pronase (Roche, Cat No 165 921)

Pronase is a recombinant protease manufactured by Roche Diagnostics,Germany, used for the dissociation of adherent avian stem cells.

Trypsine EDTA (Cambrex, Cat No BE17-161E)

Trypsine is used for the dissociation of STO cells and at late passagesfor the dissociation of avian cells adapted to Serum Free Medium. Thisenzyme of porcine origin is manufactured aseptically according to cGMPreferential conditions by a validated sterile filtration method andtested according to current E.P. The raw material, irradiated prior toformulation, is tested for porcine parvovirus in strict compliance with9/CFR 113.53.

Trypzean (Sigma, Cat No T3449)

Trypzean solution is formulated with a recombinant bovine trypsin,expressed in corn and manufactured by Sigma Aldrich utilizingProdiGene's proprietary transgenic plant protein expression system. Thisproduct is optimized for cell dissociation in both serum free andserum-supplemented adherent cell cultures.

Non Enzymatic Cell Dissociation Solution (Sigma, Cat No C5914)

This agent of dissociation is a ready to use formulation used to gentlydetach cells from the growing surface of the culture vessel. The formulacontains no protein, and allows dislodging of cells without use ofenzymes. Cellular proteins are preserved making possible immunochemicalstudies that are dependent upon the recognition of cell surfaceproteins. This enzyme was used to detach cell before FACS analysis ofbiological markers like EMA-1 (Epithelial Membrane Antigen 1) and SSEA1(Stage Specific Embryonic antigen-1).

3.2—Process of Establishment of Duck EBx Cell Line EB66

Around 360 Fertilized duck eggs were opened, the yolk were separatedfrom the albumen during the opening. The embryos were removed from theyolk with the aid of a small absorbent filter paper (Whatmann 3M paper),cut out beforehand in the form of a perforated ring with the aid of apunch. The diameter of the perforation is about 5 mm. These small ringswere sterilized using dry heat for about 30 minutes in an oven. Inpractice, during the step of embryo collection, a small paper ring isdeposited on the surface of the yolk and centered on the embryo which isthus surrounded by the paper ring. The latter is then cut out with theaid of small pairs of scissors and the whole removed is placed in aPetri dish, filled with PBS. The embryo thus carried away by the ringwere cleaned of the excess yolk in the medium and the embryonic disk,thus free of the excess vitellin, were collected with a Pasteur pipette.

The duck embryos were placed in 50 mL tubes containing PBS 1×. The duckembryos were then mechanically dissociated, washed with PBS, and seededon an inactivated layer of feeder STO cells into complete culture mediumat 39° C., 7.5% CO₂. The feeder cells were seeded in 6 well plates ordishes at around 2.7×10⁴ cell/cm². The complete culture medium iscomposed of serum free medium DMEM-Ham F12 supplemented with 10% fetalbovine serum, with IGF1, CNTF, at a final concentration of 1 ng/ml, andwith 1% non-essential amino acids, with 1% of mixture of vitamins ofcommercial origin, with sodium pyruvate at a final concentration of 0.1mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM,glutamine at a final concentration of 2.1 mM, penicillin at a finalconcentration of 100 U/ml, streptomycin at a final concentration of 100μg/ml and yeastolate 1×. Rapidly at the passage 4, the mixture ofantibiotics is no longer added to the medium.

The duck ES cells were cultured in the DMEM-Ham F12 medium up to passage4. After passage 4, the base medium is modified and DMEM-Ham F12complete medium is replaced by the SFM GTM-3 medium supplemented with10% fetal bovine serum, with IGF1, CNTF, at a final concentration of 1ng/ml, with 1% non-essential amino acids, with 1% of mixture of vitaminsof commercial origin, with sodium pyruvate at a final concentration of0.1 mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM,glutamine at a final concentration of 2.1 mM and yeastolate 1×. The duckES cells were further cultured during 14 passages in this new medium ofculture, then growth factors deprivation was performed at passage 18.IGF1 and CNTF were simultaneously removed from the medium, thus frompassage 19 to passage 24, the medium of culture was GTM-3 mediumsupplemented with 10% FBS, with 1% non-essential amino acids, with 1% ofmixture of vitamins of commercial origin, with sodium pyruvate at afinal concentration of 0.1 mM, with beta-mercapto-ethanol at a finalconcentration of 0.5 mM, glutamine at a final concentration of 2.1 mMand yeastolate 1×.

When the duck ES cells from Peking duck embryos are passaged from aculture dish to another, the seeding of culture dish was performed witharound between 7×10⁴/cm² to 12×10⁴/cm² of duck ES cells in the completeculture medium.

Then, after passage 24, depletion of feeder cells were performed by aprogressive decrease of feeder cells concentration over severalpassages. The dishes were originally seeded with around 2.7×10⁴ feedercells/cm², then around 1.8×10⁴ feeder cells/cm² between passage 25 and31, then around 1.4×10⁴ cells/cm² between passage 32 and 35, then around1×10⁴ feeder cells/cm² between passage 36 and 41, then around 0.7×10⁴feeder cells/cm² between passage 42 and 44, and finally from passage 45dishes were seeded only with avian cells and without feeder cells. Atthe end of the feeder depletion, the dishes are seeded with 9×10⁴ aviancells/cm² to 12.7×10⁴ avian cells/cm². The depletion of feeder cellsstarted at passage 25 and ended at passage 45. During the depletion offeeder cells, the duck ES cells are seeded in culture dishes at a higherconcentration than in step a), about around 9×10⁴ cell/cm² to 12.7×10⁴cell/cm² After several passages without feeder cells, growth parameters(Population Doubling Time (PDT) and Density) are studied to confirm cellstability and robustness and to initiate the deprivation of amino acids,vitamins, beta mercaptoethanol, sodium pyruvate and yeastolate. Cellsare considered as enough robust to be submitted to such deprivation if,PDT is lower than around 40 hours and cell density higher than around26×10⁴ cells/cm².

In the case of the present duck EBx® cells development, named EB66,deprivation of vitamins, sodium pyruvate, non essential amino acids andbeta mercaptoethanol were initiated at passage 52. All those additiveswere removed simultaneously from the medium. Thus, between passage 52and passage 59, the medium of culture is SFM GTM-3 supplemented withglutamine, yeastolate and FBS.

Following a short period of adaptation to the new conditions of culture,temperature decreasing was initiated. This decrease was performedprogressively between passage 60 and passage 67. After passage 67 cellswere able to grow at 37° C. After passage 67, the base medium GTM-3 wasreplaced by a new SFM base medium called Excell 65788. So, after passage67 the culture medium was Excell 65788 supplemented with 10% FBS, 2.5 mMglutamine and 1× yeastolate. At passage 80, 4×10⁶ cells were transferredin a Ultra Low Attachment (ULA) dish maintained under constant agitationto initiate anchorage-independent cells growth. To promote the growth assuspension, the base medium was modified and percentage of serum wasdecreased from 10% to 5% for the seeding in the ULA dish. Thus frompassage 80 to passage 85 the medium of culture was SFM GTM-3supplemented with 5% FBS, 2.5 mM glutamine and 1× yeastolate. Slowdecrease of FBS was initiated on EB66 cell suspension after passage 85.Serum depletion was performed by a progressive decreasing starting from2.5% serum, then 1.5% of serum concentration in SFM cell culture mediumto finally reach 0% serum in SFM cell culture medium. Serum depletionstarted at passage 86 and ended at passage 94. At the end of serumdepletion, anchorage independent dEB66 cells were able to grow at 37° C.in absence of grow factors, in absence of feeder cells, in serum freemedium.

Alter the obtaining of EB66 duck cells that are able to grow at 37° C.in the SFM GTM-3 supplemented by 2.5 mM glutamine, some furtheradaptation to SFM media were made by dilution or progressive adaptationin new SFM formulations as Excell 63066, Excell 66444, Excell CHO ACFfor example. The subcloning of suspension duck EB66 cell could alsorealized in presence or absence of yeastolate

Example 4 Duck EBx Cell Line EB26 4.1—Raw Material Duck Eggs, FeederCells, Additives, Buffers and Fixators, Cryopreservative Agents, FetalCalf Serum & Dissociating Agents (Idem as Example 3).

Duck eggs from Peking strains GL30 were used.

Medium

-   -   Medium EX-CELL 65319, 63066 and 66444 (SAFC, customized medium)    -   Medium GTM-3 (Sigma, Cat no G9916)    -   DMEM (Cambrex, Cat no BE 12-614F)

Factors

Six different recombinant factors were used:

-   -   Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech        Inc, Cat no 450-13)    -   Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc,        Cat no 100-11)    -   Recombinant Human Interleukin 6 (IL6) (Peprotech Inc, Cat no        200-06)    -   Recombinant Human soluble Interleukin 6 receptor (slL6r)        (Peprotech Inc, Cat no 200-06 R)    -   Recombinant Human Stem Cell Factor (SCF) (Peprotech Inc, Cat no        300-07)    -   Recombinant Human basic Fibroblast Growth Factor (bFGF)        (Peprotech Inc, Cat no 100-18B)

All those factors, excepted IL6r, are produced in E. Coli bacteria.Soluble IL6r is expressed in transfected HEK293 cells.

4.2—Process of Establishment of Duck EBx Cell Line EB26

The duck embryos were collected as previously described with EB66. Theduck embryos were placed in 50 mL tubes containing PBS 1×. The duckembryos were then mechanically dissociated, washed in PBS, and seeded onan inactivated layer of feeder STO cells into complete culture medium at39° C., 7.5% CO₂. The feeder cells were seeded in 6 well plates ordishes at around 2.7×10⁴ cell/cm². The complete culture medium iscomposed of serum free medium GTM-3 supplemented with 5% fetal bovineserum, with IGF1, CNTF, 11-6, 11-6R, SCF and FGF at a finalconcentration of 1 ng/ml, and with 1% non-essential amino acids, with 1%of mixture of vitamins of commercial origin, with sodium pyruvate at afinal concentration of 0.1 mM, with beta-mercapto-ethanol at a finalconcentration of 0.5 mM, glutamine at a final concentration of 2.1 mM,penicillin at a final concentration of 100 U/ml, streptomycin at a finalconcentration of 100 μg/ml and yeastolate 1×. Rapidly after the firstpassages of the cells, the mixture of antibiotics is no longer added tothe medium. The expression rapidly is understood to mean alter the first3 to 9 passages in general. The duck ES cells were cultured in thecomplete medium up to passage 9. After passage 9, the complete medium ispartially depleted in factors. Thus, between passage 10 and 13, SCF,IL6, IL6r and bFGF were removed for the medium and only recombinant IGF1and CNTF were maintained at a concentration of 1 ng/mL. A simultaneousdecease of concentration of IGF1 and CNTF is secondly performed betweenpassage 13 and 16 to finally obtain cells able to grow withoutrecombinant factors at passage 17. The factor depletion were made by aprogressive adaptation to lower concentrations of factors. When the duckES cells from Pekin duck embryos were passaged from a culture dish toanother, the seeding of culture dish was performed with around between7×10⁴/cm² to 12×10⁴/cm² of duck ES cells in the complete culture medium.Preferably, the seeding is made with around 7.3×10⁴/cm² (4×10⁶ cells/55cm² or 4×10⁶ cells/100 mm dish). After depletion of recombinant factors,a decrease of yeastolate were performed at passage 23 reaching the finalconcentration at 0.5×. Then, after passage 31, depletion of feeder cellswere performed by a progressive decrease of feeder cells concentrationover several passages. The dishes were originally seeded with around2.7×10⁴ feeder cells/cm², then around 1.8×10⁴ feeder cells/cm² betweenpassage 32 and 38, then around 1.4×10⁴ cells/cm² between passage 39 and44, then around 1×10⁴ feeder cells/cm² between passage 45 and 47, thenaround 0.7×10⁴ feeder cells/cm² between passage 48 and 50, and finallyfrom passage 51 dishes were seeded only with avian cells and withoutfeeder cells. At the end of the feeder depletion, the dishes are seededwith 9×10⁴ avian cells/cm² to 12.7×10⁴ avian cells/cm². The depletion offeeder cells started at passage 32 and ended at passage 51. During thedepletion of feeder cells, the duck ES cells are seeded in culturedishes at a higher concentration than in step a), about around 9×10⁴cell/cm² to 12.7×10⁴ cell/cm². After several passages without feedercells, growth parameters (Population Doubling Time (PDT) and Density)were studied to confirm cell stability and robustness and to initiatethe cell growth as suspension. Cells are considered as enough robust tobe submitted to a culture in suspension if, PDT is lower than around 40hours and cell density higher than around 26×10⁴ cells/cm². Moreover,cells morphology should be: round, refringent, very small and the cellsshall not attached to the plastic dish too much.

In the case of the EB26 cell development, culture in suspension wereinitiated at passage 53. 7×10⁶ cells were transferred in a Ultra Lowattachment dish and maintained under constant agitation at around 50 to70 rpm. For the next passages, cells were seeded in T175 flasks(Sarsted, ref 831812502) at a concentration comprise between 0.4 to0.5×10⁶ cells/mL. Following a short period of adaptation to the newconditions of culture, cells PDT decreased from around 160 H to 40hours. Regarding this good evolution, at passage 59, a new set ofdeprivation was performed. Thus vitamins, sodium pyruvate,beta-mercaptoethanol and non essential amino acids were removed. Thusafter passage 59, the culture medium was supplemented with 5% FBS, 0.5×yeastolate and 2.5 mM glutamine only. The serum depletion is performedon cell suspensions already depleted in growth factor, feeder cells,vitamins, non essential amino acids, sodium pyruvate andbeta-mercaptoethanol. Serum depletion was performed by a progressivedecreasing starting from 5% serum, then 2.5%, then 1.5%, of serumconcentration in SFM cell culture medium to finally reach 0% serum inSFM cell culture medium. Serum depletion started at passage 61 and endedat passage 79. At the end of serum depletion, anchorage independent duckEB26 cells were able to grow at 39° C. in absence of grow factors, inabsence of feeder cells, in serum free medium. EB26 cells were thenadapted to growth in absence of 0.5× yeastolate at 37° C. by decreasingcell culture temperature at passage 80.

After the obtaining of EB26 cells that are able to grow at 370 in theSFM GTM-3 supplemented by 2.5 mM glutamine, some further adaptation weremade by dilution or progressive adaptation on new SFM formulations asExcell 63066, Excell 66444, Excell CHO ACF. The subcloning of suspensionduck EB26 cell could also realized in presence or absence of yeastolate.

Example 5 Duck EBx Cell Line EB24 5.1—Raw Material Duck Eggs, FeederCells, Additives, Buffers and Fixators, Cryopreservative Agents, FetalCalf Serum & Dissociating Agents (Idem as Example 3).

Duck eggs from Peking strains GL30 were used.

Medium

-   -   Medium EX-CELL™ 65319, 63066 and 66444 (SAFC, customized medium)    -   Medium GTM-3 (Sigma, Cat no G9916)    -   DMEM F12 (Cambrex, Cat no BE04-687)    -   DMEM (Cambrex, Cat no BE 12-614F)

Factors

Six different recombinant factors were used:

-   -   Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech        Inc, Cat no 450-13)    -   Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc,        Cat no 100-11)    -   Recombinant Human Interleukin 6 (IL6) (Peprotech Inc, Cat no        200-06)    -   Recombinant Human soluble Interleukin 6 receptor (sIL6r)        (Peprotech Inc, Cat no 200-06 R)    -   Recombinant Human Stem Cell Factor (SCF) (Peprotech Inc, Cat no        300-07)    -   Recombinant Human basic Fibroblast Growth Factor (bFGF)        (Peprotech Inc, Cat no 100-18B)

All those factors, excepted IL6r, are produced in E. Coli bacteria.Soluble IL6r is expressed in transfected HEK293 cells.

5.2—Process of Establishment of Duck EBx® Cell Line EB24

The duck embryos were collected as previously described with EB66. Theduck embryos were placed in 50 mL tubes containing PBS 1×. The duckembryos are then mechanically dissociated and seeded on an inactivatedlayer of feeder STO cells into complete culture medium at 39° C., 7.5%CO₂. The feeder cells were seeded in 6 well plates or dishes at around2.7×10⁴ cell/cm². The complete culture medium is composed of serum freemedium DMEM-Ham F12 supplemented with 10% fetal bovine serum, with IGF1,CNTF, 11-6, II-6R, SCF and FGF at a final concentration of Ing/ml, andwith 1% non-essential amino acids, with 1% of mixture of vitamins ofcommercial origin, with sodium pyruvate at a final concentration of 0.1mM, with beta-mercapto-ethanol at a final concentration of 0.5 mM,glutamine at a final concentration of 2.1 mM, penicillin at a finalconcentration of 100 U/ml, streptomycin at a final concentration of 100μg/ml and 1× yeastolate. Rapidly after the first passages of the cells,the mixture of antibiotics is no longer added to the medium. Theexpression rapidly is understood to mean after the first 3 to 9 passagesin general.

The duck ES cells are cultured in the DMEM-Ham F12 complete medium up topassage 7. After passage 7, the base medium is modified and DMEM-Ham F12complete medium is replaced by the GTM-3 complete medium supplementedwith 10% fetal bovine serum, with IGF1, CNTF, 11-6, II-6R, SCF and FGFat a final concentration of ing/ml, with 1% non-essential amino acids,with 1% of mixture of vitamins of commercial origin, with sodiumpyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanolat a final concentration of 0.5 mM, glutamine at a final concentrationof 2.1 mM, penicillin at a final concentration of 100 U/ml, streptomycinat a final concentration of 100 μg/ml and yeastolate 1×. Thus, atpassage 11, the serum concentration is decreased at 5% and SCF, IL6,IL6r and bFGF are removed for the medium. So, from passage 11, themedium is composed of 5% FBS, with IGF1 and CNTF at a finalconcentration of 1 ng/mL with 1% non-essential amino acids, with 1% ofmixture of vitamins of commercial origin, with sodium pyruvate at afinal concentration of 0.1 mM, with beta-mercapto-ethanol at a finalconcentration of 0.5 mM, glutamine at a final concentration of 2.1 mM,penicillin at a final concentration of 100 U/ml, streptomycin at a finalconcentration of 100 μg/ml and yeastolate 1×. A simultaneous withdrawalof IGF1 and CNTF is performed at passage 22. No recombinant factors arepresent in the GTM-3 culture medium after passage 22. Duck cells weremaintained in a such medium between passage 23 and passage 28.

When the duck ES cells from Pekin duck embryos are passaged from aculture dish to another, the seeding of culture dish was performed witharound between 7×10⁴/cm² to 12×10⁴/cm² of duck ES cells in the completeculture medium. Preferably, the seeding is made with around 7.3×10⁴/cm²(4×10⁶ cells/55 cm² or 4×10⁶ cells/100 mm dish). Then, after passage 28,depletion of feeder cells is performed by a progressive decrease offeeder cells concentration over several passages. The dishes wereoriginally seeded with around 2.7×10⁴ feeder cells/cm², then around1.8×10⁴ feeder cells/cm² between passage 29 and 33, then around 1.4×10⁴cells/cm² between passage 34 and 37, then around 1×10⁴ feeder cells/cm²between passage 38 and 42, then around 0.7×10⁴ feeder cells/cm² betweenpassage 43 and 46, and finally from passage 47 dishes were seeded onlywith avian cells and without feeder cells. At the end of the feederdepletion, the dishes are seeded with 9×10⁴ avian cells/cm² to 12.7×10⁴avian cells/cm². The depletion of feeder cells started at passage 29 andended at passage 47. During the depletion of feeder cells, the duck EScells are seeded in culture dishes at a higher concentration than instep a), about around 9×10⁴ cell/cm² to 12.7×10⁴ cell/cm². After severalpassages without feeder cells, growth parameters (Population DoublingTime (PDT) and Density) were studied to confirm cell stability androbustness and to initiate the cell growth as suspension. Cells areconsidered as enough robust to be submitted to a culture in suspensionif, PDT is lower than around 40 hours and cell density higher thanaround 26×10⁴ cells/cm². Moreover, cells morphology should be: round,refringent, very small and the cells shall not attached to the plasticdish too much. In the case of the EB24 cell development, culture insuspension is initiated at passage 48. 8×10⁶ cells were transferred in aUltra Low attachment dish and maintained under constant agitation ataround 50 to 70 rpm. For the next passages, cells were seeded in T175flasks (Sarsted, ref 831812502) at a concentration comprise between 0.4to 0.5×10⁶ cells/mL. Following a short period of adaptation to the newconditions of culture, cell PDT decreased from around 248 H to 128 hoursand the next step of deprivation is then performed. Thus at passage 52,vitamines, non essential amino acids, sodium pyruvate and betamercaptethanol are removed. Regarding the good evolution of the PDTreaching 44 hours, at passage 56, from passage 57, the serum deprivationwas initiated. Thus from passage 57, the culture medium GTM-3 wassupplemented with 5% FBS, 1× yeastolate and 2.5 mM glutamine only. Theserum depletion is performed on cell suspensions already depleted ingrowth factors, feeder cells, vitamins, non essential amino acids,sodium pyruvate and beta-mercaptoethanol. Serum depletion was performedby a progressive decreasing starting from 5% serum, then 2.5%, then 2%,then 1.5% of serum concentration in SFM cell culture medium to finallyreach 0% serum in SFM cell culture medium. Serum depletion started atpassage 57 and ended at passage 77. During this serum depletion,adaptation to growth at 37° C. was also performed. Thus at passage 65,cells growing in the culture medium supplemented with 2.5% FBS weretransferred at 37° C. avoiding a progressive temperature shift. At theend of serum depletion, anchorage independent duck EB24 cells were ableto grow at 37° C. in absence of grow factors, in absence of feedercells, in serum free medium.

After the obtaining of duck EB24 cells able to grow at 37° C. in the SFMGTM-3 supplemented by 2.5 mM glutamine, some further adaptation weremade by dilution or progressive adaptation in new SFM formulations asExcell 63066, Excell 66444, Excell CHO ACF. The subcloning of suspensionduck EB24 were performed, an duck EB24-12 subclone were selected becauseof its good performance to efficiently replicate viruses.

Example 6 SPF Duck Muscovy EBx Cell Line 6.1—Raw Material Duck Eggs:

Duck SPF eggs from Muscovy strains were obtained from Le Couvoir deCerveloup (France). Those SPF duck eggs, are produced from a flocktested intensively to various poultry pathogens. Disease tested include:Salmonella gallinarum-pullorum, Mycoplasma synoviae, Mycoplasmameleagridis, Mycoplasma galliepticum, Marek's disease virus, AvianInfluenza, Type 2 Paramyxovirus, Type 3 Paramyxovirus, Newcastledisease, Type 3 Adenovirus (EDS), Gumboro disease, Avian reovirus,Reticuloendotheliosis virus, Avian encephalomyelitis, infectiousrhinotracheitis virus and Chlamydiosis. Muscovy duck eggs were onlysubmitted to a disinfection with the decontaminant to avoid any risk ofcontamination linked to the manipulation of eggs during the transport.

Feeder Cells (See Previous Examples) Media

-   -   Medium EX-CELL™ 66444 (SAFC, customized medium)    -   Medium GTM-3 (Sigma, Cat no G9916)    -   DMEM-HamF12 (Cambrex, Cat no BE04-687)

Additives

-   -   Glutamine (Cambrex, Cat no BE17-605E)    -   Pencillin/streptomycin (Cambrex, Cat no BE17-602E))    -   Non essential Amino Acids (Cambrex, Cat no BE13-114E)    -   Sodium pyruvate (Cambrex, Cat no BE13-115)    -   Vitamines (Cambrex, Cat no 13-607C)    -   Beta Mercapto Ethanol (Sigma, Cat no M7522)    -   Yeastolate (SAFC, Cat no 58902C)

Buffer and Fixators:

-   -   PBS 1× (Cambrex, Cat no BE 17-516F)

Cryoprotective Agent

-   -   Dimethyl Sulfoxyde (DMSO) (Sigma, Cat no D2650)

Factors

Two different recombinant factors were used:

-   -   Recombinant Human Ciliary Neurotrophic Factor (CNTF) (Peprotech        Inc, Cat no 450-13)    -   Recombinant Human Insulin Like Factor I (IGF1) (Peprotech Inc,        Cat no 100-11)

Those 2 factors are produced in E. Coli bacteria.

Fetal Bovine Serum

Non Irradiated Fetal Bovin Serum (FBS) (JRH, Cat No 12003)

The non irradiated serum used in the program was collected and producedin Australia. Animals used for collection were USDA inspected andacceptable for slaughter. It was added in the medium during avian stemcells culture. This batch was not submitted to irradiation to avoid thedestruction of critical proteins or components identified as essentialfor the maintenance of stem cells in culture.

Irradiated Serum (JRH, Cat No 12007)

The irradiated batch used in this program was collected in Australia.This irradiated batch was added as supplement in the DMEM medium usedfor the culture of STO cells (feeder cells). Those cells do not requireas stem cells a specific quality of serum for growth and maintenance inculture. To minimize high concentration of serum in the medium we haveadapted the STO cells to grow in presence of 4% of FBS only.

Dissociating Agents:

-   -   Pronase (Roche, Cat no 165 921)    -   Trypzean (Sigma, cat no T3449)

6.2—Process of Establishment of Muscovy Duck EBx Cell Line

Embryos from 20 fertilized SPF eggs from Muscovy ducks were collectedaccording to the process described in Example 3. The duck embryos wereplaced in 50 mL tubes containing PBS 1×. The duck embryos were thenmechanically dissociated, washed with PBS, and seeded in a well of a 12well plate coated with an inactivated layer of feeder STO cells. DuckEmbryonic cells were seeded into complete culture medium and transferredat 39° C., 7.5%5% CO₂. The feeder cells were seeded at around 2.7×10⁴cell/cm². The complete culture medium used is composed of DMEM-Ham F12supplemented with 10% fetal bovine serum, with IGF1, CNTF, at a finalconcentration of 1 ng/ml, and with 1% non-essential amino acids, with 1%of mixture of vitamins of commercial origin, with sodium pyruvate at afinal concentration of 0.1 mM, with beta-mercapto-ethanol at a finalconcentration of 0.5 mM, glutamine at a final concentration of 2.1 mM,penicillin at a final concentration of 100 U/ml, streptomycin at a finalconcentration of 100 μg/ml and yeastolate 1×. At passage 2, theDMEM-HamF12 base medium is replaced by GTM-3 base medium. The mixture ofantibiotics is no longer added to the medium after passage 4.

The duck ES cells were cultured in the complete GTM-3 medium up topassage 8. After passage 8, concentration of IGF1 and CNTF are reducedto 0.5 ng/mL. The duck ES cells were further cultured during 2 passagesin this new medium of culture, then growth factor deprivation wasperformed at passage 10. IGF1 and CNTF were simultaneously removed fromthe medium.

Thus from passage 10 to passage 37, the medium of culture was GTM-3medium supplemented with 10% FBS, with 1% non-essential amino acids,with 1% of mixture of vitamins of commercial origin, with sodiumpyruvate at a final concentration of 0.1 mM, with beta-mercapto-ethanolat a final concentration of 0.5 mM, glutamine at a final concentrationof 2.1 mM and yeastolate 1×.

When the duck ES cells isolated from Muscovy duck embryos are passagedfrom a culture dish to another, the seeding was performed with around12×10⁴/cm² of duck ES cells in the culture medium. Some conditionedmedium can be occasionally used for cell seeding to improve cellrecovery post dissociation.

Then, after passage 37, depletion of feeder cells was performed by aprogressive decreasing of feeder cells concentration over severalpassages following the step by step process previously described.

During this phase of feeder deprivation, some cells able to grow insuspension were isolated and adapted to grow without additives and serum(FIG. 4C). Anchorage-independent Muscovy duck EBx cells express ES cellsmarkers, such as telomerase, SSEA-1 and EMEA-1 (data not shown).

Example 7 EBx Cell Lines Characterization 7.1—Chicken Valo EBv13 CellsCharacterization 7.1.1—Telomerase Activity

Telomerase detection is achieved by using the Telo TAGGG Telomerase PCRELISA developed by Roche Applied Science (Telomeric Repeat AmplificationProtocol (TRAP)—Cat. No. 11 854 666 910) according to the supplierprotocol. The Telo TAGGG Telomerase PCR ELISA allows amplification ofTelomerase-mediated elongation products combined with non radioactivedetection following an ELISA protocol. The assay is valid if absorbancevalue of the negative control is less than or equal to 0.25A_(450 nm)-A_(690 nm) and if absorbance value of the positive control ishigher than or equal to 1.5 A_(450 nm)-A_(690 nm) when using 1×10³ cellequivalents in the assay. Samples are regarded as telomerase positive ifthe difference in absorbance is higher than 0.2 A_(450 nm)-A_(690 nm)units Two controls were used: the negative control is murine fibroblasts(FED cells) and the positive controls are FGB8 cells (Embryonic Stemcells established by Vivalis from 129 SV mouse embryos) and chickenEB14-O74 cells previously established in WO 03/076601.

Results obtained are summarized on the figure No 2. EBv13 cells doexpress high level of telomerase. At passage p193 and 195, thetelomerase activity is equivalent to the one of chicken EB14-O74 cells.

7.1.2—ES Cells Biological Markers

Embryonic stem cells are characterized by the expression of biologicalmarkers expressed on the cell membrane. The expression of EMA-1(Epithelial Membrane Antigen-1) and SSEA-1 (Stage Specific EmbryonicAntigen-1) on EBv13 cells were evaluated by FACS analysis. After 10minutes of fixation with PFA 4% (Para-formaldehyde), cell samples andcontrols are rinsed and pre-incubated with monoclonal antibodiesspecific of EMA-1 or SSEA-1. A second antibody conjugated to FITC isused for detection of cells expressing the 2 biological markersselected. Samples were analyzed by flow cytometry using a FACS (FlowActivated Cell Sorter) from Coulter.

FACS analysis was done on mouse fibroblasts cells (FED cells) as anegative control, murine ES FGB8 cells as a positive control, chickenEB14-O74 cells as a positive control EBx cells and EBv13 cells. Asexpected FED cells do not express biological markers whereas FGB8 andEB14-O74 cells present an important staining, respectively of, 60.13%and 78.7 for EMA-1 and 94.45% and 95% for SSEA-1 (data not shown).Chicken valo EBv13 cells population do not present any staining for EMA1(2%) and a very light one for SSEA-1 (22%).

7.1.3—Karyotype

Karyotype analysis was performed to check the cell diploidy and theavian origin of EBv13 cells. Cells in the exponential phase of growthwere harvested and treated 2 hours by colcemid (0.02 μg/mL). Afterwashing and centrifugation, an hypotonic choc is performed on cells withKCl (0.56%) during 20 minutes. Subsequently, EBv13 cells were fixed inmethanol/acetic acid (3/1) and stored overnight at −20° C. The dayafter, metaphasis were spotted on glass, stained by a wright/giemsasolution and observed under microscope. Several series of metaphaseswere observed confirming the chicken origin of EBv13 cells. No evidenceof polyploidy is observed.

7.1.4—Influence of Cell Culture Medium Composition on the Clumps Size ofEBv13 Cells

The inventors have found that the concentration of Calcium and Magnesiumin the serum-free medium used for the EBx cells culture and infectionhave an impact on the clumps size.

FIG. 10 shows the decrease in clumps size when EBv13 cells are passedfrom a medium with a high to a low Ca2+ and Mg2+ concentration.

7.2—Duck EBx Cell Lines Characterization 7.2.1—Duck EBx Cells Morphology

Transmission Electronic Microscopy analysis of dEBx® cells wereperformed by Dr. A Rivoire (Lyon, France). Duck EBx® cells display atypical embryonic stem cells morphology (i.e high nucleo-cytoplasmicratio) that resemble the phenotype of murine embryonic stem cells andVIVALIS EB14 cells described in WO2006/108846. Duck EBx® cells are smallround cells (diameter˜10 pun) with a large nucleus and nucleolus, withshort pseudopodia extending from the plasma membrane (FIG. 4A. FIG. 4Band FIG. 4C). They are highly metabolic active with a ribosome andmitochondria rich cytoplasm. They contain numerous intracellularvacuoles, a very developed Golgi system and a granulous reticulumendoplasmic.

7.2.2—Telomerase Expression of Duck EBx® Cells

Telomerase expression during different stages of establishment of induck EBx® cells was investigated by using Roche telomerase detection kit(Telomerase OCR ELISA). Telomerase is found to be highly expressed inadherent duck EBx® cells, as well as during feeder deprivation, duringthe process of adapting duck EBx® cells to suspension and during feederdeprivation.

FIG. 5 shows that duck EB24 and EB26 express high level of telomerase,just like chicken EB14 cells. Duck EB66 also express high level oftelomerase all along cell passages. This high telomerase activity isstable in EB66 cells after adaptation in different SFM (FIG. 15).

7.2.3—Duck EBx® Cells Display No Endogenous Reverse TranscriptaseActivity

Endogenous reverse transcriptase expression was investigated by directF-PERT analysis (Lovatt et al., 1999, J. Virol. Methods, 82:185-200) inClean Cells (FRANCE). Duck EBx® cell lines, EB24 (data not shown), EB66(data not shown), EB26 and EB51, display no endogenous ReverseTranscriptase (RT) activity (FIG. 6A). RT activity were detected inchicken EB14 cells culture as well as, to a lesser extend, in chickenEmbryonic fibroblast derived from Specific Pathogen Free (SPF) chickenstrain.

The presence of endogenous retroviral particles, either replicative ornon-replicative, in the cell culture supernatant of duck and chickenEBx® cells were investigated by an ELISA assay detecting the avianleukosis major capsid antigen P27 (FIG. 6B). All Duck EBx® cell lines(EB26, EB51, EB24, EB66 . . . ), as well as chicken EBv13 do not secreteALV p27 antigen. In the opposite, chicken EB14 cells do express ALV P27antigen.

7.2.4—Duck EBx Cells do not Secrete Replicative Avian Leucosis Virus(ALV)

Co-cultivation assay of duck EBx cells with quail QT6 cell line, knownto be sensitive to endogenous and exogenous ALVs, were performed todetect the presence of endogenous replicative duck viruses. FIG. 7Adescribed the principle of QT6 co-culture. The presence of replicativevirus is detected by an ELISA assay detecting the avian leucosis majorcapsid antigen P27. The assay demonstrates that none of duck EBx cellstested secrete replicative (i.e replication competent) ALV (FIG. 7B).

7.2.5—Duck EBx Cells Express Avian and Human Influenza Virus Receptors

The detection of receptors to avian (Siaα2-3Gal) and human (Siaα2-6Gal)influenza viruses on duck EBx cells were performed by fluorescent cellsorter analysis by using digoxygenin labelled lectins (Boehringer):

-   -   Sambuca nigra (SNA) agglutinin lectin specifically binds to        Siaα2-6Gal;    -   Maackia amurensis (MAA) agglutinin lectin specifically binds to        Siaα2-3Gal.

Chicken EB14 and duck EBx cells were washed in 10 mM HEPES, 150 mM NaClpH7.5 and resuspended in the same buffer at a 5.10⁶ final concentration.Cells were incubated 30 min on ice, then for an additional 15 to 30minutes in presence of SNA or MAA. Lectin treated cells were washed in10 mM HEPES, 150 mM NaCl pH7.5, prior to incubation on ice during 15 to30 minutes with FITC-labelled anti-digoxygenin antibody. Then cells arewashed in NaCl 0.9% and FACS analyzed. Chicken EB14 and duck EBx cellsexpress cell surface receptors comprising oligosaccharides withSiaα2-6Gal and Siaα2-3Gal residues (FIG. 8).

7.2.6—Karyotype

Karyotype analysis was performed to check the cell diploidy and theavian origin of duck EB24 and EB66 cells. Cells in the exponential phaseof growth were harvested and treated 3 to 6 hours by colcemid (0.6mg/mL). After washing and centrifugation, an hypotonic choc is performedon cells with KCl (0.56%) during 20 minutes. Subsequently, duck EB24 andEB66 cells were fixed in methanollacetic acid (3/1) and stored overnightat −20° C. The day alter, metaphasis were spotted on glass, stained by awright/giemsa solution and observed under microscope.

Several series of metaphases were observed confirming the duck origin ofEBx cells. No evidence of polyploidy were observed. FIG. 16 showsdiploid karyotype of duck EBx66 cells (FIG. 16).

Example 6 Poxvirus Replication in Chicken EBv13 Cell Line

Susceptibility of EBv13 cells to infection with poxvirus wasinvestigated using a recombinant Modified Vaccinia Ankara (MVA) encodinga GFP gene (Green Fluorescent Protein).

The following protocol were used: Three days before infection. 0.4×10⁶EBv13 cells (passage 188)/mL are seeded in T175 flasks under 40 mL ofSFM Excell 65319 (SAFC) supplemented with 4 mM Glutamine. The infectionis performed at a multiplicity of infection of 10² TCID50/cell (MVA-GFPstock is at 10e9.7 TCID/ml). One hour post infection, 60 ml of freshmedium is added to the flask. The culture and the infection wereperformed at 37° C., 7.5% CO₂ and agitated at 60 rpm. Each day postinfection an aliquot of the cell suspension is collected and frozen. Atthe end of the kinetic, an evaluation of the productivity is performedfollowing the TCID50 method. Briefly, the titration of infectiousMVA-GFP viruses was performed on DF-1 cells. Cells were seeded in 96flat-bottom well plates at a density of 15×10³ cells/well in DMEM medium(Biowhittaker) supplemented with 5% foetal calf serum (FCS) (SAFC) and 2mM L-glutamine (Biowhittaker). Twenty-four hours later, cells wereinfected with ten fold serially diluted samples in DMEM and incubatedfor one week at 37° C., 5% CO₂ in a humidified atmosphere. Virusinfectivity was measured through microscopic observation of globalcytopathic effect (CPE) and UV-exposed infected cells. Then, TCID50titers were calculated according the Reed and Muench method (1938, Asimple method of estimating fifty percent endpoints. Am. J. Hyg. 27,493-97). All along the experiment cell proliferation and viability aremonitored. Chicken Valo EBv13 cells appear to be highly sensitive toMVA-GFP infection (FIGS. 3A-3B).

Example 8 Poxvirus Replication in Duck EBx Cell Lines

Susceptibility of duck EBx cells to infection with poxvirus wasinvestigated using a recombinant Modified Vaccinia Ankara encoding aGFP. The virus titration was performed as previously described forchicken EBv13 cells.

8.1—Cell Culture Method

Duck EBx cells were stored in cryovials in liquid nitrogen at −196° C.(20×10⁶ cells/vial). The cryovial is directly thawed into a +37° C.pre-warmed water bath. The cell suspension is put into a 50 ml steriletube with 30 ml pre-warmed culture medium. Alter centrifugation (5 minat 300±20 g, at room temperature), 15 ml of fresh culture medium isadded on the pellet and gently homogenised. The sample is numbered usingTrypan blue. Numeration has to be ≧20×10⁶ cells and viability has tobe >70% to guarantee a good culture.

The cell suspension is plated into a T75 cm² flask and incubate at +37°C. under an 7.5% CO₂ atmosphere on an orbital shaker at 50 rpm. Freshmedium is then added daily. The cells are then passaged to increasecells biomass to seed a 3 L-bioreactor. 320.10⁶ cells are needed toinoculate a 3 L-bioreactor. A sample is taken after gently mixing toperform a numeration using trypan blue to determine cell density. A 150mL cell mix is prepared in order to obtain a cell concentration of0.4×10⁶ cells.ml⁻¹ into the 800 ml final culture volume in thebioreactor. Prior to seed cells, the pH is set in the vessel to 7.2(because pH will be decrease by CO₂ surface injection). The pO₂ is setto 50% O₂ saturation (the mass flow controller is adjusted to 100% whichcorrespond to a maximum sparger flow rate to 50 ml.min⁻¹). At thebeginning of the process, the pH is maintained by CO₂ surface injection,later, it is controlled by addition of 7.5% NaHCO₃. The surface aerationis started with air at a flow rate of 0.3 ml.min⁻¹. Cell numeration isperformed on a routine basis.

Alter 3 days of culture, cell density should be higher than 4-5×10⁶cells.ml⁻¹. If the expected cell density is reached, the virus infectionis performed at a MOI of 10⁻⁴. The vessel temperature is set to 33° C.The virus strain is thawed on ice. The infection mix is prepared in 10ml of production medium. After inoculation of the infection mix into thebioreactor, viral adsorption is performed during 1 hour. The finalproduction medium is prepared: in 1.5 L of production medium, trypsin isadded in order to obtain a final concentration in the vessel of 0.3U.ml⁻¹ (2.3 L on the whole). The pre-warmed final production medium isthen added. Every day a sample of approximately 15 ml is collected fromthe bioreactor to perform cell numeration, cell morphology analysis andto observe CPE. The metabolites such as glutamate, glutamine, lactateand glucose are analyzed all along the culture with the BioProfile Basicsoftware. Concentration of the metabolites is adjusted if necessary. Forexample, glutamine concentration is adjusted to 2 mM if necessary. Theglucose concentration is adjusted to 2 g·L⁻¹ if necessary.

Virus titration is carried-out at the end of the experiment using allcollected samples.

8.2—Results 8.2.1—Cell Growth Kinetics of Duck EBx® Cells in a 3 LFedbatch Bioreactor

Duck EBx® cells are routinely cultured in stirred-tank bioreactor. DuckEBx® derived biomass is allowed to accumulate at 37° C. in a cell growthmedium until a cell density of 5-6.10⁶ cells/mL was reached. Then themixture is diluted from around 3 to 10 fold, and cell growth kinetic isfollowed-up over a 10 days period. In such conditions, cell density of12 to 20 million cells/ml is routinely reached around day 5 to 8. ThusDuck EBx® cells display a range of splitting ratio that goes at least upto 10 to 15 fold.

8.2.2—Influence of Cell Culture Medium Composition on the Clumps SizeDuring MVA-GFP Virus Infection of Duck EBx Cells

The inventors have found that the concentration of Calcium and Magnesiumin the serum-free medium used for the EBx cells culture and infectionmay have an impact on the clumps size. The presence of small clumps ofduck EBx cells improves virus infection and propagation, leading to highMVA virus titers (FIG. 9A).

8.2.3—MVA Virus Production in 3 L-Bioreactor

Duck EBx®-derived biomass was allowed to accumulate during cellproliferation phase in Excell 66444 growth medium. Cells were theninfected with 10⁻² TCID₅₀/cell of MVA-GFP virus and the mixture wasdiluted in Excell 66444 production medium. Following addition of freshExcell medium, cell density dropped down on day 2, and at day 4, thecell density of infected cells increased and reached 12 million cell/ml.In such conditions, the MVA-GFP productivity is high. Since at day 4post-infection, the MVA-GFP titer is around 10⁸ TCID50/ml (FIG. 9B). AMVA-GFP yield of 205 TCID50/cell was obtained in duck EBx® cells.

Example 9 Production of Influenza Virus in Duck EBx Cell Lines9.1—Materials & Methods 9.1.1—Influenza Virus Infectivity Assay (TCID50)

Titration of infectious influenza viruses was performed on MDCK cells.In brief, cells were seeded in 96 flat-bottom well plates at a densityof 3×10³ cells/well in UltraMDCK medium supplemented with 2.5 mML-glutamin. Twenty-four hours later, cells were infected with ten foldserially diluted samples in UltraMDCK containing 6 μg·mL⁻¹ trypsin-EDTAand incubated for one week at 33° C., 5% CO₂ in a humidified atmosphere.Virus replication was then tested in an HA assay using chicken red bloodcells and TCID50 titers were calculated according the Reed and Muenchmethod (1938)*._*Reed L, Muench H, 1938. A simple method of estimatingfifty percent endpoints. Am. J. Hyg. 27, 493-97.

9.1.2—Single Radial Immuno-Diffusion Assay (SRID)

The concentration of haemagglutinin in samples derived from influenzavirus infected-EB14 cells, was determined as described by Wood andcolleagues*. Briefly, glass plates were coated with an agarose gelcontaining anti-Influenza serum (recommended concentration provided byNIBSC). After the gel has set, 10 μL of appropriate dilutions of thereference and the samples were loaded in 3 mm Øpunched wells. Followinga 18-24 h incubation in a moist chamber at room temperature, plates weresoaked in 0.9% NaCl and washed in distilled water. The gel was thenpressed and dried. The plates were stained on Coomassie Brillant Bluesolution for 15 min and destained twice in a mixture of methanol andacetic acid until clearly defined stained zones became visible. Afterdrying the plates, the diameter of the stained zones surrounding antigenwells were measured in two directions at right angles. Dose-responsecurves of antigen dilutions against the surface were constructed and theresults were calculated according to standard slope-ratio assay methods.*Wood J M. Et al. “An improved single-radial-immunodiffusion techniquefor the assay of influenza haemagglutinin antigen: application forpotency determinations of inactivated whole virus and subunit vaccines”(J. Biol. Stand., 1977, 5(3):237-47).

9.1.3—Western Blot Analysis of Influenza Hemagglutinin Protein

SDS-PAGE was performed as described by Laemmli UK (1970, Cleavage ofstructural proteins during the assembly of the head of bacteriophage T4.Nature 259:680-685) in 10% polyacrylamide gel. Denaturated proteins (1%SDS, 70 mM β-mercaptoethanol) were transferred to polyvinylidenedilluoride membrane (hybond P, Amersham) by a semidry blotting procedure(Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simpleapparatus without buffer tank for rapid transfer of proteins frompolyacrylamide to nitrocellulose (J Biochem Biophys Methods 10:203-209).Blots were blocked for 1 h at room temperature with a mixture composedof 5% fat dry milkpowder in TBST suplemented with 1% FCS (SAFC). Then,the blots were incubated overnight in blocking solution supplementedwith specific polyclonal anti-HA sheep serum (1:500 (NIBSC). The blotswere washed 6 times with TBST and incubated for 1 h at room temperaturewith a hrp-conjugated rabbit anti-sheep IgG polyclonal antibody (1:5000(Rockland) in blocking solution. After 6 washes with TBST, theprotein-conjugate complex was finally revealed using chemiluminescence(ECL kit, Amersham) and films (Hyperfilm, Amersham).

9.2—Influenza Virus Infection of Duck EBx® Cells in 3 L-Bioreactor9.2.1—Materials and Equipment

Cell Thawing Material

-   -   T75 cm² flasks (Sarstedt, Cat#831813502)    -   Culture medium (serum free medium)    -   L-Glutamine 200 mM (Biowhittaker, Cat#BE17-605E)    -   Orbital agitator IKA KS260 (Fisher Bioblock, Cat#F35044)

Cell Amplification Material

-   -   T175 cm² flasks (Sarstedt, Cat#831812502)    -   Culture medium (serum free medium): Excell 65319 (JRH,        Cat#65319-1000M1687) added with 2.5 mM glutamine    -   L-Glutamin 200 mM (Biowhittaker, Cat#BE17-605E)    -   D (+) Glucose (45%) (Sigma, Cat#G8769)

Production Material

-   -   Production medium (serum free medium): Excell 65629 (JRH,        Cat#65629) supplemented with 2.5 mM glutamine    -   L-Glutamin 200 mM (Biowhittaker, Cat#BE17-605E)    -   D (+) Glucose (45%) (Sigma, Cat#G8769)    -   Trypzean 1× (Sigma, Cat#T3449)    -   7.5% bicarbonate sodium solution (Sigma, Cat#205-633-8)    -   Influenza virus strain (frozen at −80° C.)

9.2.2—Cell Culture Method (Idem as for MVA Replication—Example 7.1)

Virus titration, haemmaglutinin assays (HAU) and HA antigenquantifications (western blot. SRID) are carry out at the end of theexperiment using all collected samples.

9.3—Results

The inventors demonstrate that duck EBx cells are a reliable andefficient cell substrate for the replication of various strains A and Bof influenza virus. Influenza virus production can be performed invarious vessels, such as flasks and spinner (data not shown) andbioreactors. Reproducible and efficient fedbatch process of productionof influenza virus in 3 L and 30 L stirred tank bioreactors wereobtained by the inventors. Viral yield above 15 mg/l and up to 50 mg/lof haemagglutinin are routinely obtained in flasks and in bioreactorswith strains A and B of influenza virus (FIGS. 11 and 12).

Example 10 Newcastle Disease Virus Replication in Duck EBx Cell Lines

Susceptibility of duck EBx cells to infection with Newcastle Diseasevirus was investigated using a NDV La Sota strain.

10.1—Methods

Duck EBx® cells were grown in Excell medium (SFAC) in T175 flasks at 37°C. under 7.5% CO₂ atmosphere on an orbital shaker at 60 rpm. At day 0,cells are seeded at 0.4×10⁶ cells/mL in 40 ml fresh medium. Cell culturewas incubated at 37° C., 7.5% CO₂ under shaking (60 rpm). Cell growthkinetics were followed until cell density has reached a concentrationbetween 4×10⁶ to 6×10⁶ cells/ml (usually at day 3 post seeding). At thatpoint, cells are inoculated with NDV La Sota strain at two different MOI(10⁻³ and 10⁻⁴ TCID₅₀/cells) and incubated for one additional hour at37° C., 7.5% CO₂ under shaking (60 RPM). Then the cell culture wasdiluted with the addition of 60 mL fresh viral production medium and theincubation pursued at 37° C. and 7.5% CO₂ under shaking (60 rpm). Thecell growth and virus production kinetics were performed over 7 days. Asa source of protease, recombinant trypsin (SAFC) was added every day inthe culture medium; two concentration of trypsin (0.4 and 0.75 USP/mL)were tested. Daily aliquots were removed for cell numeration, virustitration and Western blotting analysis.

The samples were separated using 10% SDS-PAGE and blotted onto PDVFmembrane (Amersham) by the semi-dry technique. Immunodetection wasperformed using chicken polyclonal antiserum against NDV (1:2000,CHARLES RIVER laboratories), followed by Alkaline phosphatase-conjugatedrabbit anti-chicken (1:5000, SIGMA). Bound secondary antibody wasdetected using the ECL-Chemiluminescence detection system kit (ROCHE).

10.2—Results

Duck and chicken EBx cells are sensitive to and replicate NDV La Sotastrain. Titers (in TCID50/ml) of NDV produced in duck EBx® cellsincrease from day 0 to day 2 μl to reach an average of 10^(6.83)_(TCID50)/mL (FIG. 13 left panel).

Western blot analysis (FIG. 13 right Panel) showed NDV viral proteins(HN, Fo/F, NP & M) expression. The viral proteins composition of NDVvirus produced in duck EBx® cells are similar to the one obtained withNDV virus produced in chicken EBx® cells. In addition, the kinetic ofrelease for viruses produced in chicken and Duck EBx cells are similar.

Example 11 Measles Virus Replication in Duck EB66 Cells

Susceptibility of duck EB66 cells to infection with measles virus wasinvestigated using a recombinant measles virus expressing greenfluorescent protein.

11.1—Methods

EB66 cells were grown in Excell medium in T175 flasks at 37 CC under7.5% CO₂ atmosphere on an orbital shaker at 60 rpm. At day 0, cells areseeded at 0.4×10⁶ cells/mL in 40 ml fresh medium. Cell culture wasincubated at 37° C., 7.5% CO₂ under shaking (60 rpm). Cell growthkinetics were followed until cell density has reached a concentrationbetween 4×10⁶ to 6×10⁶ cells/ml (usually at day 3 post seeding). At thatpoint, cells are inoculated with recombinant measles virus at twodifferent MOI (10⁻¹ and 10⁻² TCID₅₀/cells) and incubated for oneadditional hour at 37° C., 7.5% CO₂ under shaking (60 RPM). Then thecell culture was diluted with the addition of 60 mL fresh viralproduction medium and the incubation pursued at 37° C. and 7.5% CO₂under shaking (60 rpm). The cell growth and virus production kineticswere performed over 7 days. Daily aliquots were removed for cellnumeration and virus titration.

11.2—Results

EB66 cells are sensitive to and replicate measles virus. In nonoptimised conditions, titers (in TCID50/ml) of measles produced in EB66cells reach an average of 10⁷ TCID50/mL (FIG. 14).

1.-23. (canceled)
 24. A process of production of a virus which comprisesreplicating a virus in cells of an avian cell line wherein said cellline has been obtained by a method comprising the following steps: a)isolating a duck embryo around oviposition; b) dissociating the embryoof step a) into cells; c) seeding the dissociated cells of step b) inbasal medium comprising insulin like growth factor 1 (IGF-1), ciliaryneurotrophic factor (CNTF), animal serum, and a layer of feeder cells;d) culturing the cells of step c) for at least one passage; e)withdrawing the IGF-1 and CNTF from the culture of step d); f) culturingthe cells of step e) for at least one passage; g) progressivelywithdrawing the feeder cells from the culture of step f); h) culturingthe cells of step g) for at least one passage; i) progressivelywithdrawing the animal serum from the culture medium of step h) overseveral passages; and j) adapting the cells of step i) to suspensionculture conditions such that a duck cell line capable of proliferatingin basal medium in the absence of exogenous growth factors, a feederlayer, and animal serum is obtained, wherein the duck cell line does notproduce replication-competent endogenous retrovirus particles; saidprocess of production of virus comprising the steps of: 1) proliferatingsaid cells in suspension, in a serum-free medium; 2) infecting the cellswith said virus; 3) culturing said infected cells in order to allowvirus replication; and 4) harvesting said virus.
 25. The process ofclaim 24, wherein said process further comprises an additional step offeeding the cells, wherein said feeding step consists of a periodicaddition of concentrated solutions of glutamine and D-glucose to theculture medium.
 26. The process of claim 24, wherein the cell density instep 2) is of at least 1.5 million cells/ml in a fed-batch process. 27.The process of claim 24, wherein cells are cultured in a continuousstirred tank bioreactor.
 28. The process of claim 25, wherein thefeeding occurs during steps 2) and 3) of the process.
 29. The process ofclaim 25, wherein the feeding occurs during steps 1) to 3) of theprocess.
 30. The process of claim 25, wherein the feeding occurs on adaily basis.
 31. The process of claim 24, wherein said process furthercomprises the step of adding a proteolytic enzyme in the culture mediumin conditions allowing virus propagation.
 32. The process according toclaim 24, wherein cell culture is performed at a temperature of 33° C.33. The process according to claim 25, wherein the glutamineconcentration is maintained between 1 mM to 3 mM, preferably around 2mM.
 34. The process according to claim 25, wherein the D-glucoseconcentration is maintained between 1 g/l to 10 g/l, preferably around 2to 3 g/l.
 35. The process according to claim 24, wherein the withdrawalof the growth factors IGF-1 and CNTF from the culture medium in step e)is performed simultaneously.
 36. The process according to claim 24,wherein said duck is a Pekin duck.
 37. The process according to claim24, wherein said duck is a Muscovy duck.
 38. The process according toclaim 24, wherein said duck cell line is diploid.
 39. The processaccording to claim 24, wherein said duck cell line does not producereplication competent endogenous avian leucosis (ALV-E) and/orendogenous avian virus (EAV) retroviral particles.
 40. The processaccording to claim 24, wherein the genome of said duck cell line doesnot contain a proviral sequence of an avian leucosis (ALV-E) and/or anendogenous avian virus (EAV) susceptible to produce replicationcompetent endogenous retroviral particles.
 41. The process according toclaim 24, wherein said virus is selected from the group comprisingpoxviruses, recombinant poxvirus, orthomyxoviruses, recombinantorthomyxovirus, reassorted orthomyxovirus, paramyxoviruses, recombinantparamyxovirus, herpes viruses, hepadnaviruses, adenoviruses,parvoviruses, recombinant parvoviruses, reoviruses, circoviruses,coronaviruses, flaviviruses, togaviruses, birnaviruses and retroviruses.42. The process according to claim 41, wherein the poxvirus or therecombinant poxvirus is selected from the group consisting of ModifiedVaccinia Ankara (MVA) virus, Lister-Elstree vaccinia virus, LC16m8vaccinia virus, CVI78 vaccinia virus, Fowl pox virus, canary pox virus,ALVAC, NYVAC, juncopox virus, mynah pox virus, pigeonpox virus,psittacine pox virus, quail pox virus, sparrow poxvirus, starling poxvirus, and Turkey pox virus; the paramyxovirus or the recombinantparamyxovirus is selected from the group consisting of measles virus,mumps virus, rubella virus, Sendai virus, Respiratory Syncythial virus(RSV), human para-influenza types I and III, Rinderpest virus, caninedistemper virus, Newcastle disease virus, and duck para-influenza virus;the orthomyxovirus, the recombinant orthomyxovirus, or the reassortedorthomyxovirus is selected from the group consisting of human influenzavirus, avian influenza virus, swine influenza virus, equine influenzavirus, and feline influenza virus; the togavirus is selected from thegroup consisting of Sinbis virus, Semliki forest virus, O'nyong'nyongvirus, Chikungunya virus, Mayaro virus, Ross river virus, Eastern equineencephalitis virus, Western Equine encephalitis virus, Venezuelan Equineencephalitis virus, and a recombinant togavirus thereof; the retrovirusis selected from the group consisting of reticulo-endotheliosis virus,duck infectious anemia virus, duck spleen necrosis virus, and arecombinant retrovirus thereof; the parvovirus or recombinant parvovirusis a duck parvovirus; the adenovirus is selected from the groupconsisting of fowl adenovirus, goose adenovirus, duck adenovirus andpigeon adenovirus and a recombinant adenovirus thereof; the birnavirusis Infectious Bursal Disease virus; or the flavivirus is selected fromthe group consisting of Dengue virus, Japanese encephalitis virus andWest Nile virus.